Caspase inhibitors and uses thereof

ABSTRACT

The present invention relates to novel classes of compounds of formula I which are caspase and TNF-alpha inhibitors. This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are particularly well suited for inhibiting caspase and TNF-alpha activity and consequently, can be advantageously used as agents against caspase-, interleukin-1-(“IL-1”), apoptosis-, interferon-γ inducing factor- (IGIF), interferon-γ- (“IFN-γ”), or TNF-alpha mediated diseases, including inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, and degenerative diseases. This invention also relates to processes for preparing the compounds of this invention. This invention also relates to methods for inhibiting caspase and TNF-alpha activity and decreasing IGIF production and IFN-γ production and methods for treating caspase-, interleukin-1, apoptosis-, and interferon-γ-, and TNF-alpha mediated diseases using the compounds and compositions of this invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/285,051, filed Apr. 19, 2001, the content of which isincorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of medicinal chemistry and relatesto novel compounds, and pharmaceutical compositions thereof, thatinhibit caspases and/or TNF-alpha that mediate cell apoptosis andinflammation and inhibit pathophysiologic effects of excessive amountsof TNF-alpha in a mammal. The invention also relates to processes forpreparing and methods of using the compounds and pharmaceuticalcompositions of this invention to treat diseases where caspase and/orTNF-alpha activity is implicated.

BACKGROUND OF THE INVENTION

[0003] Apoptosis, or programmed cell death, is a principal mechanism bywhich organisms eliminate unwanted cells. The deregulation of apoptosis,either excessive apoptosis or the failure to undergo it, has beenimplicated in a number of diseases such as cancer, acute inflammatoryand autoimmune disorders, ischemic diseases and certainneurodegenerative disorders (see generally Science, 1998, 281,1283-1312; and Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).

[0004] Caspases are a family of cysteine protease enzymes that are keymediators in the signaling pathways for apoptosis and cell disassembly(Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathwaysvary depending on cell type and stimulus, but all apoptosis pathwaysappear to converge at a common effector pathway leading to proteolysisof key proteins. Caspases are involved in both the effector phase of thesignaling pathway and further upstream at its initiation. The upstreamcaspases involved in initiation events become activated and in turnactivate other caspases that are involved in the later phases ofapoptosis.

[0005] Caspase-1, the first identified caspase, is also known asinterleukin converting enzyme or “ICE.”Caspase-1 converts precursorinterleukin-1β (“pIL-1β”) to the pro-inflammatory active form byspecific cleavage of pIL-1β between Asp-116 and Ala-117. Besidescaspase-1 there are also eleven other known human caspases, all of whichcleave specifically at aspartyl residues. They are also observed to havestringent requirements for at least four amino acid residues on theN-terminal side of the cleavage site.

[0006] The caspases have been classified into three groups depending onthe amino acid sequence that is preferred or primarily recognized. Thegroup of caspases, which includes caspases 1, 4, and 5, has been shownto prefer hydrophobic aromatic amino acids at position 4 on theN-terminal side of the cleavage site. Another group which includescaspases 2, 3 and 7, recognizes aspartyl residues at both positions 1and 4 on the N-terminal side of the cleavage site, and preferably asequence of Asp-Glu-X-Asp. A third group, which includes caspases 6, 8,9 and 10, tolerates many amino acids in the primary recognitionsequence, but seems to prefer residues with branched, aliphatic sidechains such as valine and leucine at position 4.

[0007] The caspases have also been grouped according to their perceivedfunction. The first subfamily consists of caspases-1 (ICE), 4, and 5.These caspases have been shown to be involved in pro-inflammatorycytokine processing and therefore play an important role ininflammation. Caspase-1, the most studied enzyme of this class,activates the IL-1β precursor by proteolytic cleavage. This enzymetherefore plays a key role in the inflammatory response. Caspase-1 isalso involved in the processing of interferon gamma inducing factor(IGIF or IL-18) which stimulates the production of interferon gamma, akey immunoregulator that modulates antigen presentation, T-cellactivation and cell adhesion.

[0008] The remaining caspases make up the second and third subfamilies.These enzymes are of central importance in the intracellular signalingpathways leading to apoptosis. One subfamily consists of the enzymesinvolved in initiating events in the apoptotic pathway, includingtransduction of signals from the plasma membrane. Members of thissubfamily include caspases-2, 8, 9 and 10. The other subfamily,consisting of the effector capsases 3, 6 and 7, is involved in the finaldownstream cleavage events that result in the systematic breakdown anddeath of the cell by apoptosis. Caspases involved in the upstream signaltransduction activate the downstream caspases, which then disable DNArepair mechanisms, fragment DNA, dismantle the cell cytoskeleton andfinally fragment the cell.

[0009] Knowledge of the four amino acid sequence primarily recognized bythe caspases has been used to design caspase inhibitors. Reversibletetrapeptide inhibitors have been prepared having the structureCH₃CO—[P4]-[P3]-[P2]-CH(R)CH₂CO₂H where P2 to P4 represent an optimalamino acid recognition sequence and R is an aldehyde, nitrile or ketonecapable of binding to the caspase cysteine sulfhydryl. Rano andThornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med.Chem. Lett. 3, 2689-2692 (1993); and Nicholson et al., Nature 376, 37-43(1995). Irreversible inhibitors based on the analogous tetrapeptiderecognition sequence have been prepared where R is anacyloxymethylketone (—COCH₂OCOR″), wherein R′ is exemplified by anoptionally substituted phenyl such as 2,6-dichlorobenzoyloxy, and whereR is COCH₂X wherein X is a leaving group such as F and Cl. Thornberry etal., Biochemistry 33, 3934 (1994); and Dolle et al., J. Med. Chem. 37,563-564 (1994).

[0010] The utility of caspase inhibitors to treat a variety of mammaliandisease states associated with an increase in cellular apoptosis hasbeen demonstrated using peptidic caspase inhibitors. For example, inrodent models, caspase inhibitors have been shown to reduce infarct sizeand inhibit cardiomyocyte apoptosis after myocardial infarction, toreduce lesion volume and neurological deficit resulting from stroke, toreduce post-traumatic apoptosis and neurological deficit in traumaticbrain injury, to effectively treat fulminant liver destruction, and toimprove survival after endotoxic shock. Yaoita et al., Circulation, 97,276 (1998); Endres et al., J Cerebral Blood Flow and Metabolism, 18,238, (1998); Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlevet al., J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med.,184, 2067 (1996); and Grobmyer et al., Mol. Med., 5, 585 (1999).

[0011] In general, the peptidic inhibitors described above are verypotent against some of the caspase enzymes. However, this potency hasnot always been reflected in cellular models of apoptosis. In additionpeptide inhibitors are typically characterized by undesirablepharmacological properties such as poor oral absorption, poor stabilityand rapid metabolism. Plattner and Norbeck, in Drug DiscoveryTechnologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England,1990).

[0012] Recognizing the need to improve the pharmacological properties ofthe peptidic caspase inhibitors, peptidomimetic and non-natural aminoacid peptide inhibitors have been reported.

[0013] WO 96/40647 discloses ICE inhibitors of the formula:

[0014] wherein B is H or an N-terminal blocking group; R₁ is the aminoacid side chain of the P₁ amino acid residue wherein the P₁ amino acidis Asp; P_(n) is an amino acid residue or a heterocyclic replacement ofthe amino acid wherein the heterocycle is defined in the application; R₄is hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl; m is 0 or apositive integer; and X is N, S, O, or CH₂.

[0015] WO 97/22619 discloses inhibitors of interleukin-1β convertingenzyme of the formula:

[0016] wherein X¹ is —CH; g is 0 or 1; each J is independently selectedfrom the group consisting of —H, —OH, and —F, provided that when a firstand second J are bound to a C and said first J is —OH, said second J is—H; m is 0, 1, or 2; T is, inter alia, —CO₂H; R₁ is

[0017] where each Z is independently CO or SO₂; R₃ is as defined in theapplication; each X is independently selected from the group consistingof ═N— and ═CH—; and R₂₀ is chosen from a group containing

[0018] WO 98/16502 discloses aspartate ester inhibitors ofinterleukin-1β converting enzyme of the formula:

[0019] wherein R¹ is, inter alia, R⁵N(R^(a))CHR⁶CO—; R² is certaingroups; R⁶ is H, C₁₋₆ alkyl, —(CH₂)_(n)aryl, —(CH₂)_(n)CO₂R^(a),hydroxyl substituted C₁₋₆ alkyl, or imidazole substituted C₁₋₆ alkyl;each R^(a) is independently hydrogen, C₁₋₆ alkyl or (CH₂)_(n)aryl; andR⁵ is, inter alia, CONR^(a)R^(a).

[0020] WO 99/18781 discloses dipeptide apoptosis inhibitors having theformula:

[0021] where R₁ is an N-terminal protecting group; AA is a residue ofany natural α-amino acid, or β-amino acid; R₂ is H or CH₂R₄ where R₄ isan electronegative LG such as F, Cl, TsO—, MeO—, ArO—, ArCOO—, ArN— andArS—; and R₃ is alkyl or H.

[0022] WO 99/047154 discloses dipeptide apoptosis inhibitors having theformula:

[0023] where R₁ is an N-terminal protecting group; AA is any non-naturalamino acid or amino acid residue; and R₂ is an optionally substitutedalkyl or H as defined in the application.

[0024] WO 00/023421 discloses (substituted) acyl dipeptide apoptosisinhibitors having the formula:

[0025] where n is 0, 1, or 2; q is 1 or 2; A is a residue of any naturalor non-natural amino acid; B is a hydrogen atom, a deuterium atom, C1-10straight chain or branched alkyl, cycloalkyl, phenyl, substitutedphenyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted2-oxazolyl, (CH₂)_(m) cycloalkyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substitutedphenyl), (CH₂)_(m)(1- or 2-naphthyl), (CH₂)_(m)heteroaryl, halomethyl,CO₂R₁₃, CONR₁₄R₁₅, CH₂ZR₁₆, CH₂OCOaryl, CH₂OCO(substituted aryl),CH₂OCO(heteroaryl), CH₂OCO(substituted heteroaryl), or CH₂OPO(R₁₇)R₁₈,where R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈ and m are as defined in theapplication; R₂ is selected from a group consisting of hydrogen, alkyl,cycloalkyl, phenyl, substituted phenyl, and (CH₂)_(m)NH₂; R₃ ishydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, orsubstituted phenylalkyl; X is CH₂, C═O, O, S, NH, C(═O)NH or CH₂OCONH;and Z is an oxygen or a sulfur atom.

[0026] WO 00/061542 discloses dipeptide apoptosis inhibitors having theformula:

[0027] where R₁ is an optionally substituted alkyl or hydrogen group; R₂is hydrogen or optionally substituted alkyl; Y is a residue of a naturalor non-natural amino acid; R₃ is an alkyl, saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; and X is O, S, NR₄, or (CR₄R₅)_(n) where R₄ andR₅ are, at each occurrence, independently selected from the groupconsisting of hydrogen, alkyl and cycloalkyl, and n is 0, 1, 2, or 3; orX is NR₄, and R₃ and R₄ are taken together with the nitrogen atom towhich they are attached to form a saturated heterocyclic, partiallysaturated heterocyclic or heteroaryl group, wherein said group isoptionally substituted; or X is CR₄R₅, and R₃ and R₄ are taken togetherwith the carbon atom to which they are attached to form a saturatedcarbocyclic, partially saturated carbocyclic, aryl, saturatedheterocyclic, partially saturated heterocyclic or oxygen-containingheteroaryl group, wherein said group is optionally substituted; andprovided that when X is O, then R₃ is not unsubstituted benzyl ort-butyl; and when X is CH₂, then R₃ is not H.

[0028] Generally, the term tumor necrosis factor (TNF) refers to twoclosely related cytokines (encoded by separate genes) known as tumornecrosis factor-alpha (TNF, cachectin) and tumor necrosis factor-beta(lymphotoxin, TNF-beta). Both cytokines interact with the same cellmembrane receptors, and both have been implicated as pathogenicmediators of human illness.

[0029] TNF-alpha participates in the signaling pathways that regulatecell apoptosis and inflammation. TNF-alpha is also known as TNFSF2, TNFAand DIF. TNF-alpha is a pro-inflammatory mammalian protein capable ofinducing cellular effects by virtue of its interaction with specificcellular receptors. It is produced primarily by activated monocytes andmacrophages. Lipopoly-sacccharide (LPS, also called endotoxin), derivedfrom the cell wall of gram negative bacteria, is a potent stimulator ofTNF-alpha synthesis.

[0030] Due to the deleterious effects which can result from anover-production or an unregulated production of TNF-alpha, considerableefforts have been made to regulate the serum level of TNF-alpha. Thepathology of a number of diseases are affected by TNF-alpha, including,restinosis, inflammatory diseases of the central nervous system,demyelinating diseases of the nervous system, multiple sclerosis, septicarthritis, aneurysmal aortic disease, traumatic joint injury, peridontaldisease, macular degeneration, diabetic retinopathy, occularinflammation, keratoconus, Sjogren's syndrome, corneal graft rejection,cachexia, and anorexia.

[0031] While a number of caspase and TNF-alpha inhibitors have beenreported, it is not clear whether they possess the appropriatepharmacological properties to be therapeutically useful. Therefore,there is a continued need for small molecule caspase and TNF-alphainhibitors that are potent, stable, and have good penetration throughmembranes to provide effective inhibition of apoptosis in vivo. Suchcompounds would be extremely useful in treating the aforementioneddisease states where caspase enzymes and/or TNF-alpha cytokines play arole.

SUMMARY OF THE INVENTION

[0032] It has now been found that compounds of this invention andpharmaceutical compositions thereof are particularly effective asinhibitors of caspases, regulators of TNF-alpha levels or activity andinhibitors of cellular apoptosis and inflammatory responses. Thesecompounds have the general formula I:

[0033] wherein:

[0034] R¹ is hydrogen, CN, CHN₂, R, or —CH₂Y;

[0035] R is an aliphatic group, a substituted aliphatic group, an arylgroup, a substituted aryl group, an aralkyl group, a substituted aralkylgroup, a non-aromatic heterocyclic group, or a substituted non-aromaticheterocyclic group;

[0036] Y is an electronegative leaving group, —OR, —SR, —OC═O(R), or—OPO(R³) (R⁴);

[0037] R³ and R⁴ are independently R or OR;

[0038] R² is CO₂H, CH₂CO₂H, or optionally substituted esters, amides orisosteres thereof;

[0039] A is C═O or SO₂;

[0040] X¹ is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionallysubstituted by an alkyl group, a cycloalkyl group, a (cycloalkyl)alkylgroup, an amino acid N-terminal protecting group, or COR and —CH₂ isoptionally substituted by fluorine, an alkyl group, a cycloalkyl group,a (cycloalkyl)alkyl group, an aralkyl group, an aryl group, an alkyloxygroup, an alkylthioxy group, an aryloxy group, an arylthioxy group, anoxo group (i.e., ═O), or a NHCOR group;

[0041] X² is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionallysubstituted by an alkyl group, or an amino acid N-terminal protectinggroup and —CH2 is optionally substituted by an alkyl group, an arylgroup, an alkyloxy group, an alkylthioxy group, an aryloxy group, anarylthioxy group, or an oxo (i.e., ═O) group, a NHCOR group; X¹ and X²optionally form part of a phenyl ring that is fused to the adjoiningring Q;

[0042] X³ is CH₂ or X² and X³ optionally form part of a phenyl ring thatis fused to the adjoining ring Q, provided that when X² forms a ringwith X³, then X² does not form a ring with X¹;

[0043] any two hydrogens attached to adjacent positions in ring Q areoptionally replaced by a double bond; and

[0044] Z is an optionally substituted ring selected from the groupconsisting of a carbocyclic, an aryl, a saturated heterocycle, apartially saturated heterocycle, and a heteroaryl wherein the ring isconnected to A at a ring carbon.

[0045] The compounds of this invention are potent inhibitors of caspaseand TNF activity. They have inhibiting activity across a range ofcaspase targets with good efficacy in cellular models of apoptosis andinflammation. In addition, these compounds are expected to have improvedcell penetration and pharmacokinetic properties and, as a consequence oftheir potency, have improved efficacy against diseases where caspasesand/or TNF-alpha are implicated.

[0046] The invention also relates to methods for inhibiting the releaseof TNF-alpha from various cells or decreasing TNF-alpha levels oractivity using the compounds and compositions of this invention. Theinvention also relates to methods for identifying agents useful fordecreasing TNF-alpha levels or activity and treating TNF-alpha mediateddiseases. The invention additionally relates to kits comprising acompound or composition of this invention and a tool for measuringTNF-alpha levels or activity.

DETAILED DESCRIPTION OF THE INVENTION

[0047] This invention provides novel compounds and pharmaceuticallyacceptable derivatives thereof that are particularly effective ascaspase inhibitors and/or regulators of TNF-alpha levels or activity.The invention also provides methods for using the compounds to treatcaspase and/or TNF-alpha mediated disease states in mammals. Thecompounds have the general formula I:

[0048] wherein:

[0049] R¹ is hydrogen, CN, CHN₂, R, or —CH₂Y;

[0050] R is an aliphatic group, a substituted aliphatic group, an arylgroup, a substituted aryl group, an aralkyl group, a substituted aralkylgroup, a non-aromatic heterocyclic group, or a substituted non-aromaticheterocyclic group;

[0051] Y is an electronegative leaving group, —OR, —SR, —OC═O(R), or—OPO(R³) (R⁴);

[0052] R³ and R⁴ are independently R or OR;

[0053] R² is CO₂H, CH₂CO₂H, or optionally substituted esters, amides orisosteres thereof;

[0054] A is C═O or SO₂;

[0055] X¹ is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionallysubstituted by an alkyl group, a cycloalkyl group, a (cycloalkyl)alkylgroup, an amino acid N-terminal protecting group, or COR and —CH₂ isoptionally substituted by fluorine, an alkyl group, a cycloalkyl group,a (cycloalkyl)alkyl group, an aralkyl group, an aryl group, an alkyloxygroup, an alkylthioxy group, an aryloxy group, an arylthioxy group, anoxo group (i.e., ═O), or a NHCOR group;

[0056] X² is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionallysubstituted by an alkyl group or an amino acid N-terminal protectinggroup and —CH₂ is optionally substituted by an alkyl group, an arylgroup, an alkyloxy group, an alkylthioxy group, an aryloxy group, anarylthioxy group, or an oxo (i.e., ═O) group, a NHCOR group; X¹ and X²optionally form part of a phenyl ring that is fused to the adjoiningring Q;

[0057] X³ is CH₂ or X² and X³ optionally form part of a phenyl ring thatis fused to the adjoining ring Q, provided that when X² forms a ringwith X³, then X² does not form a ring with X¹;

[0058] any two hydrogens attached to adjacent positions in ring Q areoptionally replaced by a double bond; and

[0059] Z is an optionally substituted ring selected from the groupconsisting of a carbocyclic, an aryl, a saturated heterocycle, apartially saturated heterocycle, and a heteroaryl wherein the ring isconnected to A at a ring carbon.

[0060] As used herein, the following definitions shall apply unlessotherwise indicated. The term “condition” or “state” refers to anydisease, disorder or effect that produces deleterious biologicalconsequences in a subject.

[0061] According to this invention, “TNF” or “TNF alpha” refers toTNF-alpha.

[0062] The term “subject” refers to an animal, or to one or more cellsderived from an animal. Preferably, the animal is a mammal, mostpreferably a human. The cells can be in any form, including but notlimited to cells retained in tissue, cell clusters, immortalized cells,transfected or transformed cells, and cells derived from an animal thathave been physically or phenotypically altered.

[0063] The term “patient” as used in this application refers to anymammal, preferably humans.

[0064] The term “interferon gamma inducing factor” or “IGIF” refers to afactor which is capable of stimulating the endogenous production ofIFN-γ.

[0065] The term “aliphatic” means straight chain, branched or cyclicC₁-C₁₂ hydrocarbons which are completely saturated or contain one ormore units of unsaturation. For example, suitable aliphatic groupsinclude substituted or unsubstituted linear, branched or cyclic alkyl,alkenyl, or alkynyl groups and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl and (cycloalkyl)alkenyl. The term“alkyl” used alone or as part of a group or larger moiety refers to bothstraight and branched chains containing one to twelve carbon atoms.

[0066] The term “halogen” means F, Cl, Br, or I. The term “heteroatom”means nitrogen, oxygen or sulfur and shall include any oxidized form ofnitrogen and sulfur, such as N(O), S(O), and S(O)₂ and the quaternizedform of nitrogen. It is stood that the compounds of the presentinvention are limited to those existing in nature or chemically stable.

[0067] A combination of substituents or variables is permissible only ifsuch a combination results in a stable or chemically feasible compound.A stable compound or chemically feasible compound is one that is notsubstantially altered when kept at a temperature of 40° C. or less, inthe absence of moisture or other chemically reactive conditions, for atleast a week.

[0068] The term “aryl”, used alone or as part of a group or largermoiety, refers to monocyclic or polycyclic aromatic carbon ring systems,and monocyclic or polycyclic heteroaromatic ring systems containing oneor more heteroatoms, having five to fourteen atoms. Such groups include,but are not limited to, phenyl, naphthyl, anthryl, furanyl, thienyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indolyl,isoindolyl, indolinyl, benzofuranyl, benzothiophenyl, indazolyl,benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl, quinolinyl,isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydrofuranyl, phthalimidinyl,tetrazolyl, and chromanyl.

[0069] The term “aralkyl” refers to an alkyl substituted by an arylgroup. The term “heteroaryl” refers to an aryl group containing one ormore heteroatoms. The term “heteroaralkyl” refers to an aralkyl groupcontaining one or more heteroatoms.

[0070] The term “heterocyclic group” or “heterocycle” refers tosaturated and unsaturated monocyclic or polycyclic ring systemscontaining one or more heteroatoms and with a ring size of three toeight. Such groups include, but are not restricted to, aziranyl,oxiranyl, azetidinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl,dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl,pyranyl, piperidinyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl,piperazinyl, trithianyl, quinuclidinyl, oxepanyl, and thiepanyl. Theterm “heterocyclic ring”, whether saturated or unsaturated, also refersto rings that are optionally substituted. The term “heterocyclylalkyl”refers to an alkyl group substituted by a heterocyclic ring.

[0071] The term “carbocyclic group” or “carbocyclyl” refers to saturatedor unsaturated non-aromatic monocyclic or polycyclic carbon ring systemswhich can be fused to aryl or heterocyclic groups. Examples couldinclude cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, indanyl,tetrahydronaphthyl and the like. The term “carbocyclylalkyl” refers toan alkyl substituted by a carbocyclic group.

[0072] An aryl group (including a heteroaryl) or an aralkyl (including aheteroaralkyl) group, such as benzyl or phenethyl, can contain one ormore substituents. Examples of suitable substituents of an aryl oraralkyl group include halogen, CF₃, —R⁵, —OR⁵, —OH, —SH, —SR⁵, protectedOH such as acyloxy and those described in Wuts and Greene, ProtectiveGroups in Organic Synthesis, 3^(rd) Edition John Wiley & Sons, 1999),—NO₂, —CN, —NH₂, —NHR⁵, —N(R⁵)₂, —NHCOR⁵, —NHCONHR⁵, —NHCON(R⁵)₂,—NR⁵COR⁵, —NHCO₂R⁵, —CO₂R⁵, —CO₂H, —COR⁵, —CONHR⁵, —CON(R⁵)₂, —S(O)₂R⁵,—SONH₂, —S(O)R⁵, —SO₂NHR⁵, or —NHS(O)₂R⁵, where R⁵ is an aliphatic or asubstituted aliphatic group, preferably having one to three carbons, oran aryl or a substituted aryl group, with the proviso that when R⁵ is ansubstituted aryl group, said aryl can not be substituted by ansubstituted aryl.

[0073] An aliphatic group or a non-aromatic heterocyclic ring cancontain one or more substituents. Examples of suitable substituents ofan aliphatic group or a non-aromatic heterocyclic ring include thoselisted above for an aryl or aralkyl group as well as the following: ═O,═S, ═NNHR⁶, ═NN(R₆)₂, ═N—OR⁶, ═NNHCOR⁶, ═NNHCO₂R⁶, ═NNHSO₂R⁶, and ═NR⁶,wherein R₆ is an aliphatic group or a substituted aliphatic group.

[0074] A substitutable nitrogen on an aromatic or non-aromaticheterocyclic ring can be optionally substituted. Suitable substituentson the nitrogen include R⁶, COR⁶, S(O)₂R⁶, and CO₂R⁶.

[0075] The term “electronegative leaving group” has the definition knownto those skilled in the art (see March, Advanced Organic Chemistry,4^(th) Edition, John Wiley & Sons, 1992). Examples of electronegativeleaving groups include halogens such as F, Cl, Br, and I, aryl- andalkyl-sulfonyloxy groups, trifluoro-methanesulfonyloxy, OR⁷, SR⁷,—OC═O(R⁷), —OPO(R⁸)(R⁹), where R⁷ is an aliphatic group, an aryl group,an aralkyl group, a carbocyclic group, a carbocyclylalkyl group, aheterocyclic group, or an heterocyclylalkyl group; and R⁸ and R⁹ areindependently R⁷ or OR⁷.

[0076] The term “amino acid N-terminal protecting group” has thedefinition known to those skilled in the art. Examples of amino acidN-terminal protecting groups include those described in Wuts and Greene,Protective Groups in Organic Synthesis, 3^(rd) Edition John Wiley &Sons, 1999).

[0077] Isosteres or bioisosteres of carboxylic acids and esters resultfrom the exchange of an atom or a group of atoms to create a newcompound with similar biological properties to the parent carboxylicacid or ester. The bioisosteric replacement can be physicochemically ortopologically based. An example of an isosteric replacement for acarboxylic acid is CONHSO₂J where J is an alkyl group such as methyl,ethyl, propyl, butyl, and the like.

[0078] Compounds of this invention where R² is CO₂H or CH₂CO₂H,γ-ketoacids or δ-ketoacids respectively, can exist in solution as eitherthe open form (a) or the cyclized hemiketal form (b) (y=1 forγ-ketoacids, y=2 for δ-ketoacids). The representation herein of eitherisomeric form is meant to include the other.

[0079] Likewise it will be apparent to one skilled in the art thatcertain compounds of this invention can exist in tautomeric forms orhydrated forms, all such forms being within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structures; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³ C- or ¹⁴C-enrichedcarbon are within the scope of the application.

[0080] A preferred R¹ group is CH₂Y where Y is an electronegativeleaving group, OR, SR, or —OC(═O)(R) and most preferably Y is F.

[0081] Preferably R² is CO₂H, esters, amides or isosteres thereof.

[0082] X¹ is preferably CH₂; X² is preferably CH₂; or X¹ and X² combineto form part of an optionally substituted phenyl ring fused to ring Q.More preferably X¹ and X² are both CH₂.

[0083] A is preferably CO.

[0084] Z is preferably an optionally substituted aryl which is connectedto A at a ring carbon.

[0085] Representative examples of compounds of the present invention areshown below in Table 1. TABLE 1 Representative Compounds

No. Z 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

[0086] The compounds of this invention can be prepared in general bymethods known to those skilled in the art for analogous compounds, asillustrated by the general Schemes I and II below and by the preparativeexamples that follow.

[0087] Reagents: (a) CbzOSuc/THF/TEA; (b) LiOH/THF/H₂O; (c)EDC/DMAP/HOBt; (d) H₂/10% Pd on C/EtOAc; (e) TBTU/DIPEA/DMF; (f)Dess-Martin periodinane; (g) TiCl₄/DCM; and (k) TFA/DCM.

[0088] Scheme I shows a general approach for making the presentcompounds. The starting ester hydrochloride 1 is first protected as acarbamate using a known amino acid N-protecting protocol, for exampleCbz-OSuc (benzyloxycarbonyl-O-succinimidyl) in THF (tetrahydrofuran) inthe presence of base, such as TEA (triethylamine, step a). The ester 2is then hydrolyzed using base or, when the ester is a t-butyl group,using trifluoroacetic acid (TFA). The acid 3 is then coupled with theamino alcohol 4, using for example, EDC(1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride), DMAP(4-dimethylaminopyridine) and HOBt (1-hydroxybenzo-triazole), to provide5. Depending on the nature of R¹ and R², an amino ketone can be used inplace of 4, which avoids the subsequent oxidation step. In the case offluoromethyl ketones where R¹ is CH₂F, the amino alcohol 4 can beobtained according to the method of Revesz et al., Tetrahedron Lett.,1994, 35, 9693. The carbamate 5 is then deprotected using catalytichydrogenation, for example, H₂ with Pd on C in EtOAc (ethyl acetate), oracidolysis. The amine 6 is then N-substituted with the desired acylatingor sulfonylating agent using standard methods as shown, for example, bystep e, TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate) in DMF (dimethylformamide) in the presence of DIPEA(diisopropylethylamine). The hydroxyl group in compound 7 is thenoxidized by standard methods as shown, for example, by step f. Finally,compound 8 is treated appropriately according to the nature of R² togenerate I, using TiCl₄ in DCM (dichloromethane) or TFA in DCM. Forexample, if I requires R² to be a carboxylic acid, then R² in 4 ispreferably an ester and the final step in the scheme is hydrolysis.

[0089] Reagents: (h) DIPEA/DMF/TBTU/^({circle over (Z)})—CO₂H; (l)

[0090] DIPEA/DCM/^({circle over (Z)})—COCl; (i) LiOH/THF/H₂O; (m)KOH/MeOH/H₂O; (j) EDC/DMAP/HOBt; (f) Dess-Martin periodinane; (g)TiCl₄/DCM; and (k) TFA/DCM.

[0091] Scheme II represents an alternative approach for making thepresent compounds. The starting ester hydrochloride 1 is first reactedwith either a carboxylic acid or an acid chloride using known amide bondforming reactions. The amide 9 is then hydrolyzed using base. The acid10 is then coupled with the amino alcohol 4 to provide 11. Depending onthe nature of R¹ and R², an amino ketone can be used in place of 4,which avoids the subsequent oxidation step. In the case of fluoromethylketones where R¹ is CH₂F, the amino alcohol 4 can be obtained accordingto the method of Revesz et al., Tetrahedron Lett., 1994, 35, 9693. Thehydroxyl group in compound 11 is then oxidized by standard methods asshown, for example, by step f. Finally compound 12 is treatedappropriately according to the nature of R² to generate I. For example,if I requires R² to be a carboxylic acid, then R² in 4 is preferably anester and the final step in the scheme is hydrolysis.

[0092] Certain compounds of this invention can be obtained as follows.The parent heterocyclic esters 1 or their acids or derivatives used inscheme I are either commercially available or can be prepared usingstandard methods. For example, the parent heterocyclic ester 1 where X¹are each CH₂ is commercially available (H-homoproline-OMe). The parentheterocyclic ester 1, where X¹ is CH₂; and X² is oxygen, can be preparedby standard methods (Wolfe et al., Tetrahedron Lett., 1979, 3913). Theparent heterocyclic ester 1, where X¹ is CH₂ and X² is sulfur, can beprepared by standard methods (Miyazaki et al., Bull. Chem. Soc. Jpn.,1993, 66, 536). The parent heterocyclic ester 1, where X² is CH₂; and X¹is oxygen, can be prepared by standard methods (Kogami et al., Bull.Chem. Soc. Jpn., 1987, 60, 2963; Asher et al., Tetrahedron Lett., 1981,141; and Brown et al., J. Chem. Soc. Perkin Trans. 1,1985, 2577). TheFmoc N-protected amino acid is also commercially available. The parentheterocyclic ester 1, where X² is CH₂; and X¹ is sulfur, can be preparedby standard methods (Kogami et al., Bull. Chem. Soc. Jpn., 1987, 60,2963; and Sakai et al., Chem. Pharm. Bull., 1981, 29, 1554). Thecorresponding free amino acid is also commercially available. The parentheterocyclic esters 1, where X¹⁻² is CH₂ and have various substituentscan be prepared by standard methods (Shuman et al., J. Org. Chem., 1990,55, 738; Agami et al., Synlett, 1997, 799; and Nazih et al., Synlett,1998, 1337).

[0093] The compounds of this invention are designed, inter alia, toinhibit caspase activity and/or decrease TNF-alpha levels or activity.These compounds can be assayed, for example, for their ability toinhibit apoptosis, inhibit the release of IL-1β, inhibit caspaseactivity, and/or decrease TNF-alpha levels or activity. Assays for eachof the activities are known in the art and are described below in detailin the Examples. Accordingly, these compounds are capable of targetingand inhibiting events in caspase- (for example, IL-1-), apoptosis-,IGIF-, IFN-γ-, and TNF-α-mediated diseases, and the ultimate activity ofthe relevant protein in inflammatory diseases, autoimmune diseases,destructive bone, proliferative disorders, infectious diseases, anddegenerative diseases.

[0094] Compounds of this invention also inhibit conversion of pro-IGIFinto active, mature IGIF by inhibiting ICE. The term “interferon gammainducing factor” or “IGIF” refers to a factor which is capable ofstimulating the endogenous production of IFN-γ.

[0095] Because ICE is essential for the production of mature IGIF(IL-18), inhibition of ICE effectively blocks initiation ofIGIF-mediated physiological effects and symptoms, by inhibitingproduction of mature IGIF. IGIF is in turn essential for the productionof IFN-γ. ICE therefore effectively blocks initiation of IFN-γ- mediatedphysiological effects and symptoms, by inhibiting production of matureIGIF and thus production of IFN-γ.

[0096] Compounds of this invention also inhibit the release of TNF-alphafrom activated cells.

[0097] The pharmaceutical compositions and methods of this invention,therefore, will be useful for controlling caspase and TNF-alpha activityin vivo. The compositions and methods of this invention will thus beuseful for controlling caspase, IL-1, IGIF, IFN-γ, or TNF-alpha levelsin vivo and for treating or reducing the advancement, severity oreffects of caspase, IL-1-, apoptosis-, IGIF-, IFN-γ-, or TNF-alphamediated conditions, including diseases, disorders or effects.

[0098] According to another embodiment, the invention provides acomposition comprising a compound of this invention or apharmaceutically acceptable derivative thereof, as described above, anda pharmaceutically acceptable carrier.

[0099] According to another embodiment, the compositions of thisinvention can further comprise another therapeutic agent. Such agentsinclude, but are not limited to, a thrombolytic agent such as tissueplasminogen activator and streptokinase, an anti-inflammatory agent, amatrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator (e.g.,bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF,methionine enkephalin, interferon alpha, diethyldithiocarbamate, tumornecrosis factor, naltrexone and rEPO), a prostaglandin, or ananti-vascular hyperproliferation compound.

[0100] The term “pharmaceutically acceptable carrier” refers to anon-toxic carrier that can be administered to a patient, together with acompound of this invention, and which does not destroy thepharmacological activity thereof.

[0101] Pharmaceutically acceptable carriers that can be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

[0102] A “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ester, salt of an ester or otherderivative of a compound of this invention which, upon administration toa recipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. The methods for preparing salts or esters of a compoundof this invention are known to one of skill in the art.

[0103] Pharmaceutically acceptable derivatives of the compounds of thisinvention include, without limitation, esters, amino acid esters,phosphate esters, metal salts and sulfonate esters.

[0104] In pharmaceutical compositions comprising only a compound offormula I as the active component, methods for administering thesecompositions can additionally comprise the step of administering to thesubject an additional agent. Such agents include, but are not limitedto, a thrombolytic agent such as tissue plasminogen activator andstreptokinase, an anti-inflammatory agent, a matrix metalloproteaseinhibitor, a lipoxygenase inhibitor, a cytokine antagonist, animmunosuppressant, an anti-cancer agent, an anti-viral agent, acytokine, a growth factor, an immunomodulator (e.g., bropirimine,anti-human alpha interferon antibody, IL-2, GM-CSF, methionineenkephalin, interferon alpha, diethyldithiocarbamate, tumor necrosisfactor, naltrexone and rEPO), a prostaglandin, or an anti-vascularhyperproliferation compound. When a second agent is used, the secondagent can be administered either as a separate dosage form or as part ofa single dosage form with the compounds or compositions of thisinvention.

[0105] The amount of compound present in the above-describedcompositions should be sufficient to cause a detectable decrease in theseverity of the disease, in caspase activity and/or cell apoptosis, orin TNF-alpha activity and/or cell apoptosis as measured by any of theassays described in the Examples.

[0106] The compounds of this invention can be employed in a conventionalmanner for controlling IGIF and IFN-γ levels in vivo and for treatingdiseases or reducing the advancement or severity of effects which aremediated by a caspase, IL-1, apoptosis, IGIF, IFN-γ or TNF-alpha. Suchmethods of treatment, their dosage levels and requirements can beselected by those of ordinary skill in the art from available methodsand techniques.

[0107] If pharmaceutically acceptable salts of the compounds of thisinvention are utilized in these compositions, those salts are preferablyderived from inorganic or organic acids and bases. Included among suchacid salts are the following: acetate, adipate, alginate, aspartate,benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate,camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth.

[0108] Also, the basic nitrogen-containing groups can be quaternizedwith such agents as lower alkyl halides, such as methyl, ethyl, propyl,and butyl chloride, bromides and iodides; dialkyl sulfates, such asdimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides suchas decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides,aralkyl halides, such as benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products are thereby obtained.

[0109] The compounds utilized in the compositions and methods of thisinvention can also be modified by appending appropriate functionalitiesto enhance selective biological properties. Such modifications are knownin the art and include those which increase biological penetration intoa given biological system (e.g., blood, lymphatic system, centralnervous system), increase oral availability, increase solubility toallow administration by injection, alter metabolism and alter rate ofexcretion.

[0110] According to a preferred embodiment, the compositions of thisinvention are formulated for pharmaceutical administration to a mammal,preferably a human being.

[0111] Such pharmaceutical compositions of the present invention can beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection andinfusion techniques. Preferably, the compositions are administeredorally or intravenously.

[0112] Sterile injectable forms of the compositions of this inventioncan be aqueous or oleaginous suspension. These suspensions can beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono-ordi-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil and castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms canalso be used for the purposes of formulation.

[0113] If a solid carrier is used, the preparation can be tableted,placed in a hard gelating capsule in powder or pellet form, or in theform of a troche or lozenge. The amount of solid carrier will vary,e.g., from about 25 mg to 400 mg. When a liquid carrier is used, thepreparation can be, e.g., in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampule or nonaqueousliquid suspension. Where the composition is in the form of a capsule,any routine encapsulation is suitable, for example, using theaforementioned carriers in a hard gelatin capsule shell.

[0114] A syrup formulation can consist of a suspension or solution ofthe compound in a liquid carrier for example, ethanol, glycerine, orwater with a flavoring or coloring agent. An aerosol preparation canconsist of a solution or suspension of the compound in a liquid carriersuch as water, ethanol or glycerine; whereas in a powder dry aerosol,the preparation can include e.g., a wetting agent.

[0115] Formulations of the present invention comprise an activeingredient together with one or more acceptable carrier(s) thereof andoptionally any other therapeutic ingredient(s). The carrier(s) should be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

[0116] The pharmaceutical compositions of this invention can be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried cornstarch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents can also be added.

[0117] Alternatively, the pharmaceutical compositions of this inventioncan be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient which is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

[0118] The pharmaceutical compositions of this invention can also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

[0119] Topical application for the lower intestinal tract can beeffected in a rectal suppository formulation (see above) or in asuitable enema formulation. Topically-transdermal patches can also beused.

[0120] For topical applications, the pharmaceutical compositions can beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

[0121] For ophthalmic use, the pharmaceutical compositions can beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticalcompositions can be formulated in an ointment such as petrolatum.

[0122] The pharmaceutical compositions of this invention can also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well known in the art of pharmaceuticalformulation and can be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents known in the art.

[0123] It will be recognized by one of skill in the art that the formand character of the pharmaceutically acceptable carrier or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration, and other well-known variables.

[0124] The above-described compounds and compositions are particularlyuseful in therapeutic applications relating to an IL-1 mediated disease,an apoptosis mediated disease, an inflammatory disease, an autoimmunedisease, a destructive bone disorder, a proliferative disorder, aninfectious disease, a degenerative disease, a skin disease, a diseaseassociated with cell death, an excess dietary alcohol intake disease, aviral mediated disease, retinal disorders, uveitis, inflammatoryperitonitis, osteoarthritis, pancreatitis, asthma, adult respiratorydistress syndrome, glomerulonephritis, rheumatoid arthritis, systemiclupus erythematosus, scleroderma, chronic thyroiditis, Grave's disease,autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmuneneutropenia, thrombocytopenia, chronic active hepatitis, myastheniagravis, inflammatory bowel disease, Crohn's disease, psoriasis, atopicdermatitis, contact dermatitis, scarring, graft vs host disease, organtransplant rejection, organ apoptosis after burn injury, osteoporosis,leukemias and related disorders, myelodysplastic syndrome, multiplemyeloma-related bone disorder, acute myelogenous leukemia, chronicmyelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiplemyeloma, haemorrhagic shock, sepsis, septic shock, burns, trauma,systemic inflammatory response syndrome, multiple organ dysfunctionsyndrome, Shigellosis, Alzheimer's disease, Parkinson's disease,Huntington's disease, Kennedy's disease, prion disease, cerebralischemia, epilepsy, myocardial ischemia, acute and chronic heartdisease, myocardial infarction, congestive heart failure,atherosclerosis, coronary artery bypass graft, spinal muscular atrophy,amyotrophic lateral sclerosis, multiple sclerosis, HIV-relatedencephalitis, aging, alopecia, neurological damage due to stroke,ulcerative colitis, traumatic brain injury, spinal chord injury,hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever,Japanese encephalitis, various forms of liver disease, renal disease,polycystic kidney disease, H. pylori-associated gastric and duodenalulcer disease, HIV infection, tuberculosis, and meningitis.

[0125] The above-described compounds and compositions are also useful intherapeutic applications relating to a TNF mediated disease. The phrase“TNF-alpha mediated disease” means, all diseases states in whichTNF-alpha plays a role, either by excessive production or release ofTNF-alpha, itself, or by TNF-alpha causing an event that triggers orexacerbates the disease, such as production or release of anotherpathophysiological biochemical agent, or cytokine. In one preferredembodiment, TNF-alpha plays a direct role.

[0126] Such TNF-alpha mediated diseases can include, e.g., restinosis,inflammatory diseases such as inflammatory diseases of the centralnervous system, demyelinating diseases of the nervous system, multiplesclerosis, septic arthritis, aneurysmal aortic disease, traumatic jointinjury, peridontal disease, macular degeneration, diabetic retinopathy,occular inflammation, keratoconus, Sjogren's syndrome, corneal graftrejection, cachexia, and anorexia.

[0127] Excessive TNF-alpha tissue levels have been implicated inmediating or exacerbating a number of diseases including: rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions; also general sepsis, gram-negative sepsis,septic shock, endotoxic shock, toxic shock syndrome, adult respiratorydistress syndrome (ARDS), cerebral malaria, chronic pulmonaryinflammatory disease, silicosis, asbestosis, pulmonary sarcoidosis, boneresorption diseases, graft vs. host reactions, allograft rejections;also fever and myalgias due to bacterial or viral infections, such asinfluenza; cachexia secondary to acquired immune deficiency syndrome(AIDS), keloid formation, scar tissue formation, Crohns disease,ulcerative colitis, or pyresis; a number of “autoimmune diseases” suchas multiple sclerosis, autoimmune diabetes, and systemic lupuserythematosus.

[0128] TNF-alpha inhibitors are useful in the treatment of a variety ofallergic, traumatic and other injurious disorders including: asthma,chronic bronchitis, atopic dermatitis, urticaria, allergic rhinitis,allergic conjunctivitis, eosiniophilic granuloma, ulcerative colitis,Crohn's disease, reperfusion injury of the myocardium and brain, chronicglomerulonephritis, and adult respiratory distress syndrome (ARDS).

[0129] The compounds of this invention can inhibit the release ofTNF-alpha and thus can be useful for inhibiting or blocking severalpathophysiological effects of TNF-alpha at injury or surgery sites andthus also inhibit the release of other pathophysiological biochemicalproducts from cells such as histamines, prostaglandins, bradykinins, andperoxidases.

[0130] As discussed above, TNF-alpha inhibitors can be very effective inthe treatment of disorders which follow cellular, tissue or organ injuryor surgery, and can be as effective, or even more potent, thancorticosteroids or immunosuppressants without producing the side effectscommon to these agents.

[0131] This invention also relates to a therapeutic method of (1)inhibiting TNF-alpha release from cells and (2) preventing the untoward,toxic or lethal effects of excessively high tissue levels of TNF-alphain a mammal, including a human. This method comprises administering to amammal an effective TNF-alpha inhibiting quantity of one or more of theabove compounds. This method also can be used for the prophylactictreatment or prevention of certain TNF-alpha mediated or exacerbateddiseases amenable thereto. The invention provides a method for thetreatment of allergic, traumatic, radiation, chemical, microbial andother injurious disorders by administering to a mammal, including ahuman, in need thereof an effective amount of such compounds.

[0132] The compounds, by inhibiting or blocking the release of TNF-alphaor decreasing TNF-alpha levels and activity, as well as thepathophysiologic actions of excessive levels of TNF-alpha in each ofthese circumstances, directly facilitate the arrest or resolution of thetissue or organ damage, and facilitates the restoration of normalfunction. Together, these actions relate their novel use in treatingtissue trauma, or other injury disorders caused by infection, allergy,immunologic phenomena, burns, radiation exposure, neoplastic disease,toxic chemicals and expressed as cardiovascular damage, neurologicinjury, renal damage, liver damage, pancreatic damage, as well asascites, localized edema, dermal damage and dermal blister.

[0133] The term “inhibiting the release of TNF-a” means:

[0134] a) decrease of in vivo TNF-alpha levels in a mammal such as ahuman; or

[0135] b) a down regulation of TNF-alpha levels in vitro or in vivo; or

[0136] c) a down regulation of TNF-alpha activity, by inhibition of thedirect synthesis of TNF-alpha or a post-translation event.

[0137] The compounds can be useful in inhibiting the release ofTNF-alpha by monocytes, macrophages, neuronal cells, endothelial cells,epidermal cells, mesenchymal cells (for example: fibroblasts, skeletalmyocytes, smooth muscle myocytes, cardiac myocytes) and many other typesof cells.

[0138] The term “condition” or “state” refers to any disease, disorderor effect that produces deleterious biological consequences in asubject.

[0139] The level of TNF-alpha protein in the blood or cell of a patientor a cell culture (i.e., in the cells and/or in the culture media) canbe determined by assaying for immunospecific binding to TNF-alpha or toproteins that are known to be produced as a result of the presence ofactive TNF-alpha. Such assays are known in the art. For example, theimmunoassays which can be used include, but are not limited, tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, FACS analysis and protein Aimmunoassays. Such assays are well known in the art (see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety).

[0140] Competitive binding assays can also be used to determine thelevel of TNF-alpha. One example of a competitive binding assay is aradioimmunoassay comprising the incubation of labeled proteins fromcells expressing TNF-alpha(e.g., ³H or ¹²⁵I) with a TNF-alpha antibodyin the presence of increasing amounts of unlabeled TNF-alpha, and thedetection of the TNF-alpha antibody bound to the labeled TNF-alpha.

[0141] TNF-alpha levels can also be assayed by activity assays known inthe art. For example, samples of treated cell cultures or from bloodfrom patients can be used in TNF-alpha activity assays known in the art,e.g., J. Immunol. Methods, 1995, 178, 71-76; Burns, 1994, 20(1), 40-44.

[0142] Dosage levels of between about 0.01 and about 100 mg/kg bodyweight per day, preferably between 0.5 and about 75 mg/kg body weightper day and most preferably between about 1 and 50 mg/kg body weight perday of the active ingredient compound are useful in a monotherapy.

[0143] Typically, the pharmaceutical compositions of this invention willbe administered from about 1 to 5 times per day or alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that can be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

[0144] When the compositions of this invention comprise a combination ofa compound of formula I and one or more additional therapeutic agents,both the compound and the additional agent should be present at dosagelevels of between about 10% to 80% of the dosage normally administeredin a monotherapy regime.

[0145] Upon improvement of a patient's condition, a maintenance dose ofa compound, composition or combination of this invention can beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, can be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients can, however, require intermittent treatment on a long-termbasis upon any recurrence or disease symptoms.

[0146] It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of active ingredients will also depend upon the particularcompound and other therapeutic agent, if present, in the composition.

[0147] One embodiment of this invention provides a method for treatingor preventing an IL-1- mediated disease or an apoptosis-mediated diseasein a subject comprising the step of administering to the subject anycompound, pharmaceutical composition, or combination described herein.

[0148] Another embodiment of this invention provides a method forinhibiting a caspase-mediated function in a subject comprising the stepof administering to the subject any compound, pharmaceuticalcomposition, or combination described.

[0149] Another embodiment of this invention provides a method fordecreasing IGIF or IFN-γ production in a subject comprising the step ofadministering to the subject any compound, pharmaceutical composition,or combination described.

[0150] Another embodiment of this invention provides a method fortreating complications associated with coronary artery bypass grafts ina subject comprising the step of administering to the subject anycompound, pharmaceutical composition, or combination described herein.

[0151] Another embodiment of this invention provides a method forpreserving cells comprising the step of bathing the cells in a solutionof any compound described herein. Such method using caspase inhibitorshas been reported [Schierle et al., Nature Medicine, 5, p. 97 (1999);and Natori et al., Transplantation, 68, pp. 89-96 (1999)]. The amount ofcaspase inhibitor needed will depend on the effectiveness of theinhibitor for a given cell type and the length of time required topreserve cells from apoptotic cell death.

[0152] Another embodiment of this invention provides a method forpreserving cells needed for an organ transplant or for preserving bloodproducts, using any compound, pharmaceutical composition, or combinationdescribed herein. Li et al., Transfusion, 40, pp. 1320-1329 (2000).

[0153] Another embodiment of this invention provides a method fortreating various forms of cancer in a subject comprising the step ofadministering to the subject any compound, pharmaceutical composition,or combination described herein as a component of immunotherapy. Droinet al., Oncogene, 16, pp. 2885-2894 (1998); Boudard et al., Leukemia,14, pp. 2045-2051 (2000); Faderl et al., Clinical Cancer Research, 5,pp. 4041-4047 (1999); Ozoren et al., Cancer Research, 60, pp. 6259-6265(2000); Sasaki et al., British Journal of Urology, 81, pp. 852-855(1998); and Hedlund et al., Prostate, 36, pp. 92-101 (1998).

[0154] In a preferred embodiment, the invention provides a method oftreating a mammal, having one of the aforementioned diseases, comprisingthe step of administering to said mammal a pharmaceutically acceptablecomposition described above. In this embodiment, if the patient is alsoadministered another therapeutic agent or caspase inhibitor, it can bedelivered together with the compound of this invention in a singledosage form, or, as a separate dosage form. When administered as aseparate dosage form, the other caspase inhibitor or agent can beadministered prior to, at the same time as, or following administrationof a pharmaceutically acceptable composition comprising a compound ofthis invention.

[0155] A kit according to this invention comprises a compound or apharmaceutically acceptable derivative thereof or pharmaceuticalcomposition of this invention and a tool for measuring TNF alpha levelsand/or activity in vitro or in vivo. The kit can further compriseinstructions for using the contents of the kit. A tool for measuringTNF-alpha levels of this invention refer to materials that can be usedto measure the TNF gene product (i.e., RNA or protein) or activity. Suchmethods are described for example above. Thus, a tool according to thisinvention can include e.g., an anti-TNF antibody, a TNF-alpha DNA probeor a genetically engineered cell line responsive to TNF alpha levelsdescribed above.

[0156] The methods for identifying a compound or composition thatdecreases TNF-alpha activity and/or levels according to this inventioninclude methods for screening of a plurality of compounds orcompositions for their ability to decrease TNF-alpha activity and/orlevels. For example, high-throughput screening is a desired embodimentof this invention. According to one embodiment of this invention,high-throughput screening can be achieved by having cells in culture ina plurality of wells in a microtiter plate, adding a different compoundor composition to each well and comparing the TNF-alpha levels and/oractivity in each cell culture to the TNF-alpha levels or activitypresent in a cell culture in a control well. Controls that are usefulfor the comparison step according to this invention include cells orsubjects that have not been treated with a compound or composition andcells or subjects have been treated with a compound or composition thatis known to have no effect on TNF-alpha levels or activity. According toone embodiment of this invention, the high throughput screening isautomated so that the steps including the addition of the cells to theplate up to the data collection and analysis after addition of thecompound or composition are done by machine. Instruments that are usefulin the comparison step of this invention, e.g., instruments that candetect labeled objects (e.g., radiolabelled, fluorescent or coloredobjects) or objects that are themselves detectable, are commerciallyavailable and/or known in the art. Accordingly, compounds andcompositions according to this invention that are useful for decreasingTNF-alpha levels and/or activity can be quickly and efficientlyscreened.

[0157] In order that this invention be more fully understood, thefollowing preparative and testing examples are set forth. These examplesare for the purpose of illustration only and are not to be construed aslimiting the scope of the invention in any way.

EXAMPLES Example 1 [3S/R(2S)]-5-Fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid (Compound 1)

[0158]

[0159] Method A:(S)-1-(Benzyloxycarbonyl) 2-piperidine-carboxylic AcidMethyl Ester

[0160] A stirred suspension of (S) -Piperidine-carboxylic acid methylester hydrochloride (2.00 g, 11.13 mmol) in anhydrous THF (40 ml) atroom temperature was treated with triethylamine (3.41 ml, 24.50 mmol).The reaction mixture was stirred at room temperature for 30 min beforethe addition of N-(benzyloxycarbonyloxy)succinimide (3.05 g, 12.23mmol). The resulting mixture was stirred for 2 hr, before being dilutedwith ethyl acetate (20 ml), washed with 2 N HCl, saturated aq. NaHCO₃,saturated aq. NaCl, dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by flash chromatography (20% ethyl acetate inhexane) to afford the sub-title compound as a colourless oil (2.0085 g,65%): ¹H NMR (400 MHz, CDCl₃) δ1.19-1.1.54 (2H, m), 1.58-1.80 (4H, m),2.18-2.33 (1H, m), 2.90-3.15 (1H, m), 3.66-3.81 (3H, m), 4.03-4.21 (1H,m), 4.81-5.25 3H, m), 7.28-7.45 (5H, m).

[0161] Method B: (S)-1-(Benzyloxycarbonyl)-2-piperidine-carboxylic Acid

[0162] A stirred solution of(S)-1-(benzyloxy-carbonyl)-2-piperidinecarboxylic acid methyl ester(2.00 g, 7.21 mmol) in THF (20 ml) at room temperature was treated withwater (10 ml). Lithium hydroxide (190 mg, 7.93 mmol) was added and theresulting mixture stirred at room temperature for 3 hr. An additionalquantity of lithium hydroxide (40 mg, 1.67 mmol) was added and theresulting mixture was stirred for 2 hr prior to the removal of theorganic solvent. The resulting solution was washed with diethyl etherand the remaining aqueous layer was made acidic with 2 N HCl prior to asecond extraction step with ethyl acetate. The organic layer was thenrecovered, dried (Na₂SO₄), filtered and concentrated to reveal acolorless oil (1.9927 g, 105%) which crystallized upon standing: ¹H NMR(400 MHz, CDCl₃) δ 1.30-1.88 (5H, m), 2.22-2.41 (1H, m), 3.00-3.21 (1H,m), 4.08-4.25 (1H, m), 4.91-5.30 (3H, m), 7.27-7.48 (5H, m).

[0163] Method C: [3S/R, 4S/R,(2S)]-5-Fluoro-4-hydroxy-3-[1-(benzyloxycarbonyl)-2-piperidinecarboxamido]-pentanoicAcid Tert-Butyl Ester

[0164] A stirred mixture of (S)-1-(benzyloxycarbonyl)-2-piperidinecarboxylic acid (4.82 g, 18.31 mmol),3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester (3.99 g,19.25 mmol), HOBt (2.72 g, 20.13 mmol), DMAP (2.57 g, 21.04 mmol) andanhydrous THF (60 ml) was cooled to 0° C. before EDC (3.86 g, 20.13mmol) was added. The mixture was allowed to warm to room temperatureover 16 hrs before being concentrated under reduced pressure. Theresidue was purified by flash chromatography (60% ethyl acetate inhexane) to afford the sub-title compound as a white foam (7.3754 g,72%): ¹H NMR (400 MHz, CDCl₃) δ 1.31-1.80 (14H, m), 2.20-2.38 (1H, m),2.49-3.07 (3H, m), 3.11-3.70 (1H, m), 3.80-4.58 (4H, m), 4.70-5.28 (1H,m), 6.58-7.05 (1H, m), 7.23-7.48 (5H, m)

[0165] Method D: [3S/R, 4S/R,(2S)]-5-Fluoro-4-hydroxy-3-[2-piperidinecarboxamido]-pentanoic AcidTert-Butyl Ester

[0166] A stirred solution of [3S/R, 4S/R,(2S)]-5-fluoro-4-hydroxy-3-[1-(benzyloxycarbonyl)-2-piperidinecarboxamido]-pentanoicacid tert-butyl ester (7.37 g, 16.29 mmol) in ethyl acetate (150 ml) wastreated with 10% Pd/C (830 mg). The reaction mixture was then thoroughlydegassed and placed under a hydrogen balloon. The resulting mixture wasstirred at room temperature for 3 hrs after which it was filteredthrough celite and concentrated to the sub-title compound as a colorlessgum (5.17 g, 100%): ¹H NMR (400 MHz, CDCl₃) δ 1.16-2.00 (15H, m),2.51-2.78 (3H, m), 2.99-3.09 (1H, m), 3.18-3.28 (1H, m), 3.93-4.56 (4H,m), 7.39-7.58 (1H, m); ¹⁹F (376 MHz, CDCl₃) δ −229.34 (t), −229.42 (t),−229.87 (t), 230.02 (t).

[0167] Method E: [3S/R, 4S/R,(2S)]-5-Fluoro-4-hydroxy-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidine-carboxamido]-pentanoicAcid Tert-Butyl Ester

[0168] A stirred solution of [3S/R, 4S/R,(2S)]-5-fluoro-4-hydroxy-3-[2-piperidinecarboxamido]-pentanoic acidtert-butyl ester (520 mg, 1.63 mmol), in DMF (9.7 ml) at roomtemperature was treated with DIPEA (311 μl, 1.80 mmol). The resultingmixture was allowed to stir for 30 min before being treated with2-phenyl-thiazole-4-carboxylic acid (335 mg, 1.63 mmol) and TBTU (524mg, 1.63 mmol). The mixture was stirred at room temperature for 16 hrand then diluted with ethyl acetate. The resulting solution was washedwith 2 N HCl, saturated aq. NaHCO₃, saturated aq. NaCl, dried (Na₂SO₄),filtered and concentrated to reveal an oil. The residue was purified byflash chromatography (60% ethyl acetate in hexane) to afford thesub-title compound as a colorless oil (463 mg, 56%): ¹H NMR (400 MHz,CDCl₃) δ 1.10-1.85 (15H, m), 2.22-2.89 (3H, m), 3.09-4.78 (6H, m),5.20-5.43 (1H, m), 7.40-7.56 (3H, m), 7.81-8.11 (3H, m).

[0169] Method F: [3S/R,(2S)]-5-fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid Tert-Butyl Ester

[0170] A stirred solution of [3S/R, 4S/R,(2S)]-5-Fluoro-4-hydroxy-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoicacid tert-butyl ester (462 mg, 0.91 mmol) in anhydrous DCM (25 ml) wastreated with 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3 (1H)-one(426 mg, 1.00 mmol) at 0° C. The resulting mixture was kept at 0° C. for2 hr, diluted with DCM, and washed with saturated aq. Na₂S₂O₃.5H₂O,saturated aq. NaHCO₃, saturated aq. NaCl. dried (Na₂SO₄) andconcentrated. The residue was purified by flash chromatography (33%ethyl acetate in hexane) to afford the sub-title compound as a whitesolid (376 mg, 82%): IR (solid) 1731, 1619, 1506, 1460, 1363, 1260, 1158cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 1.16-1.81 (14H, m), 2.25-2.42 (1H, m),2.69-3.25 (3H, m), 4.48-5.46 (5H, m), 7.36-8.32 (7H, m); ¹³C NMR (100MHz, CDCl₃) δ 21.08, 21.29, 21.33, 25.24, 25.72, 27.23, 28.14, 36.60,41.33, 41.52, 46.10, 46.27, 52.06, 52.77, 52.84, 82.25, 82.40, 82.66,83.81, 85.68, 125.09, 125.49, 126.50, 126.69, 127.02, 127.10, 129.46,129.55, 131.00, 131.18, 132.85, 133.04, 133.29, 150.75, 150.92, 163.50,163.65, 165.17, 168.07, 168.14, 170.07, 170.23, 171.37, 202.87, 203.02;¹⁹F (376 MHz, CDCl₃) δ −231.36, −231.69, −231.86, −232.28; MS (LR, ES)Calculated for C₂₅H₃₀FN₃O₅S: 503.5974.

[0171] Method G: [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid

[0172] A stirred solution of [3S/R,(2S)]-5-fluoro-4-oxo-3-[1-(2-phenyl-thiazole-4-carbonyl)-2-piperidinecarboxamido]-pentanoicacid tert-butyl ester (370 mg, 0.73 mmol) in anhydrous DCM (20 ml) wastreated with a 1 M solution of titanium tetrachloride in DCM (3.67 ml,3.67 mmol) at −10° C. The resulting mixture was warmed to 0° C. and keptat this temperature for 1 hr. The reaction mixture was then diluted withDCM, and washed with 2 N HCl, saturated aq. NaCl, dried (Na₂SO₄) andconcentrated. The residue was purified by reverse phase HPLC(acetonitrile/water) to afford the title compound as a white foam (102mg, 31%): IR (solid) 1798, 1736, 1674, 1617, 1517, 1479, 1470, 1265; ¹HNMR (400 MHz, d₆-DMSO+TFA) δ 1.18-1.79 (4H, m), 2.08-2.28 (1H, m),2.42-3.50 (5H, m), 4.08-5.40 (4H, m), 7.45-8.28 (6H, m), 8.41-8.67 (1H,m); ¹³C NMR (100 MHz, d₆-DMSO+TFA) δ 19.07, 19.24, 23.29, 23.69, 25.63,25.72, 26.42, 26.49, 31.54, 33.23, 43.65, 46.14, 50.81, 50.91, 51.56,51.64, 56.26, 56.34, 80.07, 81.87, 81.97, 83.66, 83.75, 102.43, 102.47,102.63, 102.66, 125.17, 125.24, 128.19, 129.52, 131.40, 149.51, 162.07,162.61, 162.67, 165.34, 165.55, 169.71, 169.86, 170.64, 170.72, 171.75,201.84, 201.20, 201.34; ¹⁹F (376 MHz, d₆-DMSO) δ −226.74, −226.82,−226.84, −227.00, −230.37, −230.60, −230.83, −232.41, −232.55, −232.62,−232.7; MS (LR, ES) calculated for C₂₁H₂₂FN₃O₅S: 447.4890, ES−446.408,ES+448.184.

Example 2 [3S/R(2S)]-5-Fluoro-4-oxo-3-[1-(isoquinoline-1-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid, Trifluoroacetic Acid Salt (Compound 2)

[0173]

[0174] Method H: (S)-1-(Isoquinoline-1-carbonyl)-2-piperidinecarboxylicAcid Methyl Ester

[0175] A stirred solution of (S)-1-Piperidine-carboxylic acid methylester hydrochloride (1.00 g, 5.57 mmol), in DMF (20 ml) at roomtemperature was treated with DIPEA (2.12 ml, 12.25 mmol). The resultingmixture was allowed to stir for 30 min before being treated with1-isoquinolinecarboxylic acid (964 mg, 5.57 mmol) and TBTU (1.79 g, 5.57mmol). The mixture stirred at room temperature for 4 hr, diluted withethyl acetate, washed with saturated aq.NaHCO₃, saturated aq. NaCl,dried (Na₂SO₄), filtered and concentrated. The residue was purified byflash chromatography (67% ethyl acetate in hexane) to afford thesub-title compound as a colourless gum (1.05 g, 63%): ¹H NMR (400 MHz,CDCl₃) δ 1.00-2.51 (6H, m), 3.05-3.38 (2H, m), 3.60-3.95 (3H, m),4.35-4.95 (1H, m), 5.70-5.80 (1H, m), 7.55-7.95 (3H, m), 8.13-8.29 (1H,m), 8.48-8.61 (1H, m)

[0176] Method I: (S)-1-(Isoquinoline-1-carbonyl)-2-piperidinecarboxylicAcid

[0177] A stirred solution of(S)-1-(isoquinoline-1-carbonyl)-2-piperidinecarboxylic acid methyl ester(1.05 g, 3.52 mmol) in THF (20 ml) at room temperature was treated withwater (10 ml). Lithium hydroxide (84 mg, 3.51 mmol) was then added andthe resulting mixture was stirred at room temperature for 16 hrs. Theresulting mixture was concentrated to remove the organic solvent. Theresulting solution was then washed with diethyl ether and the remainingaqueous layer was made acid with 2 N HCl. The resulting solution wasextracted with ethyl acetate and the organic layer was separated, dried(Na₂SO₄), filtered and concentrated to reveal a white solid (902 mg,90%): ¹H NMR (400 MHz, d₄-MeOH) δ 1.20-2.52 (6H, m), 3.10-3.39 (2H, m),4.10-4.90 (1H, m), 7.68-8.55 (5H, m).

[0178] Method J: [3S/R, 4S/R,(2S)]-5-Fluoro-4-hydroxy-3-[1-(isoquinoline-1-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid Tert-Butyl Ester

[0179] A stirred mixture of(S)-1-(isoquinoline-1-carbonyl)-2-piperidinecarboxylic acid (278 mg,0.98 mmol), 3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester(213 mg, 1.03 mmol), HOBt (145 mg, 1.07 mmol), DMAP (137 mg, 1.12 mmol)and anhydrous THF (25 ml) was cooled to 0° C. then EDC (206 mg, 1.07mmol) was added. The mixture was allowed to warm to room temperatureduring 16 hrs then concentrated under reduced pressure. The residue waspurified by flash chromatography (5% methanol in DCM) to afford thetitle compound as a white foam (425 mg, 92%): ¹H NMR (400 MHz, CDCl₃) δ1.20-3.30 (17H, m), 3.95-4.18 (2H, m), 4.29-4.64 (4H, m), 4.86-5.01 (1H,m), 7.65-8.00 (4H, m), 8.10-9.00 (3H, m); ¹⁹F (376 MHz, CDCl₃) δ−229.39, −229.41, −229.57, −229.60, −229.64, −229.69, −230.41, −231.08.

[0180] [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(isoquinoline-1-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid Tert-Butyl Ester

[0181] This sub-title compound was prepared using procedures similar tothose described in method F as a white foam (268 mg, 63%): ¹H NMR (400MHz, CDCl₃) δ 1.20-2.60 (15H, m), 2.69-4.30 (4H, m), 4.83-5.78 (4H, m),7.23-7.35 (1H, m), 7.57-8.00 (1H, m), 8.13-8.30 (1H, m), 8.45-8.72 (1H,m), 9.08-9.73 (1H, m); ¹⁹F (376 MHz, CDCl₃) δ −231.53, −231.69, −231.70,−232.13; MS (LR, ES) calculated for C₂₅H₃₀FN₃Os: 471.5334, ES−470.327,ES+472.270.

[0182] Method K: [3S/R,(2S)]-5-Fluoro-4-hydroxy-3-[1-(isoquinoline-1-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid Trifluoroacetic Acid Salt

[0183] An ice cooled solution of trifluoroacetic acid (5 ml) inanhydrous DCM (5 ml) was added to a stirred ice cold solution of [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(isoquinoline-1-carbonyl)-2-piperidine-carboxamido]-pentanoicacid tert-butyl ester (240 mg, 0.51 mmol) in anhydrous DCM (15 ml). Themixture was stirred at 0° C. for 2 hr and 4° C. for 40 hr. The mixturewas concentrated under reduced pressure and the residue was dissolved indry DCM. This process was repeated four times in order to remove excesstrifluoroacetic acid. The gum was triturated with diethyl ether toafford the title compound as an off white solid (126 mg, 53%): IR(solid) 1794, 1736, 1646, 1441, 1250, 1198, 1150, 1055 cm⁻¹; ¹H NMR (400MHz, d₆-DMSO+TFA) δ 1.18-2.36 (6H, m), 2.59-3.45 (4H, m), 4.10-5.51 (4H,m), 7.60-8.78 (7H, m); ¹³C NMR (100 MHz, d₆-DMSO +TFA); ¹⁹F (376 MHz,d₆-DMSO) δ −226.75, −226.81, −227.00, −232.62, −232.66, −233.09; MS (LR,ES) calculated for C₂₁H₂₂FN₃O₅: 415.42, ES−414.269, ES+416.198.

Example 3 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-benzoyl-2-piperidinecarboxamido]-pentanoicAcid (Compound 3)

[0184]

[0185] Method L: (S)-1-Benzoyl-2-piperidinecarboxylic Acid Methyl Ester

[0186] A stirred suspension of (S)-Piperidine-carboxylic acid methylester hydrochloride (1.009 g, 5.65 mmol) in anhydrous DCM (7 ml) at 0°C. was treated with diisopropylamine (3 ml, 17.34 mmol) and then benzoylchloride (0.72 ml, 6.21 mmol). The resulting mixture was then stirred at0° C. for 4 hr, before being diluted with DCM. The resulting solutionwas washed with 1 N HCl, saturated aq. NaHCO₃, saturated aq. NaCl, dried(MgSO₄), filtered and concentrated to reveal an oil. The residue waspurified by flash chromatography (20% ethyl acetate in hexane) to affordthe title compound as a colourless oil (1.221 g, 87%): ¹H NMR (400 MHz,CDCl₃) δ 1.25-1.80 (5H, m), 2.10-2.39 (1H, m), 2.75-3.27 (1H, m),3.55-3.68 (0.66H, m), 3.70-3.79 (3H, m), 5.41-5.54 (0.66H, m), 5.41-5.53(0.66H, m), 7.26-7.46 (5H, m).

[0187] Method M: (S)-1-Benzoyl-2-piperidinecarboxylic Acid

[0188] A stirred solution of (S)-1-benzoyl-2-piperidinecarboxylic acidmethyl ester (1.221 g, 4.94 mmol) in a solution of methanol (5 ml) inwater (5 ml) at 0° C. was treated with potassium hydroxide (305 mg, 5.43mmol). The resulting mixture was stirred at 0° C. for 2 hr. A furtherquantity of potassium hydroxide (111 mg, 1.97 mmol) was then added andthe resulting mixture was stirred for 1.5 hr, then concentrated. Theresulting aqueous solution was then washed with DCM and the remainingaqueous layer was made acid with 1 N HCl. The resulting solution wasextracted with ethyl acetate and the organics were seperated, dried(MgSO₄), filtered and concentrated to reveal a crystalline solid (870mg, 76%): ¹H NMR (400 MHz, CDCl₃) 1.21-1.78 (5H, m), 1.98-2.27 (1H, m),2.71-3.20 (1H, m), 3.28-3.38 (3H, m), 3.41-3.53 (0.5H, m), 4.21-4.47(1H, m), 5.11-5.25 (0.5H, m), 7.27-7.39 (2H, m), 7.40-7.50 (3H, m).

[0189] The title compound was then prepared by subjecting(S)-1-Benzoyl-2-piperidinecarboxylic acid to procedures similar to thosedescribed in methods J, F and K. The product was isolated after RP-HPLC(MeCN/H₂O) as a white foam (25 mg, 10% last step): IR (solid) 3318,2944, 1787, 1736, 1675, 1611 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO) δ 1.37-1.63(5H, m), 2.05-2.18 (1H, m), 2.60-2.94 (2H, m), 3.25-3.46 (2H, m),4.34-4.77 (2H, m), 5.12-5.29 (2H, m), 7.34-7.90 (5H, m), 8.12-8.58 (1H,m); ¹³C NMR (100 MHz, d₆-DMSO) δ 20.54, 24.64, 25.11, 26.87, 27.80,34.65, 45.82, 52.42, 58.46, 83.42, 126.37, 127.20, 128.74, 129.75,129.86, 136.37, 171.38, 172.22, 173.33, 202.70, 202.82; ¹⁹F (376 MHz,d₆-DMSO) δ −226.52, −226.71, −226.84, −226.91, −230.13, −232.28,−232.39, −232.62, −232.66; MS (FAB+ve, HR) calculated for C₁₈H₂₁FN₂O₅(MH+) 365.1513, found 365.1519.

Example 4 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(2-methyl-4-trifluoromethyl-thiazole-5-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid (Compound 4)

[0190]

[0191] The title compound was prepared using procedures similar to thosedescribed in methods A-G. The product was isolated as a white foam (27.1mg, 8% last step): IR (solid) 1794, 1736, 1632, 1436, 1355, 1203, 1165,1126 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO+TFA) δ 1.14-1.74 (6H, m), 2.01-2.23(1H, m), 2.42-3.55 (7H, m), 4.06-4.80 (2H, m), 5.00-5.39 (2H, m),8.02-8.71 (1H, m); ¹³C NMR (100 MHz, d₆-DMSO+TFA) δ 18.87, 19.07, 23.25,23.85, 25.88, 31.47, 33.03, 33.20, 33.40, 44.18, 46.05, 46.16, 50.78,50.82, 50.94, 51.02, 51.14, 51.26, 51.42, 56.58, 56.94, 80.08, 81.84,81.93, 82.05, 83.73, 83.82, 102.44, 102.63, 116.52, 116.68, 116.90,120.79, 122.31, 124.80, 125.55, 125.65, 129.02, 129.24, 129.32, 133.75,135.65, 160.84, 161.00, 168.93, 169.18, 169.39, 170.64, 171.78, 172.64,200.93, 201.26, 201.40; ¹⁹F (376 MHz, d₆-DMSO) δ−61.54, −226.61,−226.76, −226.86, −227.02, −228.01, −229.32, −229.86, −230.48, −231.39,−232.37, −232.55, −232.59, −232.69; MS (LR, ES) calculated forC₁₇H₁₉F₄N₃O₅S: 453.4157, ES−452.327, ES+454.141.

Example 5 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(7-methoxy-benzofuran-2-carbonyl)-2-piperidine-carboxamido]-pentanoicAcid (Compound 5)

[0192]

[0193] The title compound was prepared using procedures similar to thosedescribed in methods A-G. The product was isolated as a white foam (24.0mg, 12% last step): IR (solid) 1794, 1736, 1627, 1589, 1427, 1269, 1203cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO+TFA) δ 1.20-1.80 (4H, m), 2.10-2.29 (1H,m), 2.52-3.66 (5H, m), 4.05-5.42 (4H, m), 6.92-7.49 (4H, m), 8.29-8.90(1H, m); ¹³C NMR (100 MHz, d₆-DMSO+TFA) δ 20.52, 20.7524.64, 25.21,27.00, 32.91, 34.61, 44.97 (CH₂), 47.73, 52.41, 52.66, 52.91, 53.25,57.77 (CH, CH₃), 83.44, 85.21 (CH₂), 103.94, 104.13 (C), 108.71, 113.92,124.78 (CH), 128.60, 143.56, 145.57, 148.54, 158.17, 158.55, 158.93,159.32, 160.59, 160.78, 170.91, 172.21, 173.28, 202.66, 202.80; ¹⁹F (376MHz, d₆-DMSO) δ −75.65, −226.85, −226.94, −228.09, −230.52, −230.83,−232.64, −232.74, −232.96.; MS (LR, ES) calculated for C₂₁H₂₃FN₂O₇:434.4251, ES−433.386, ES+435.171.

Example 6 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(3-chloro-benzo[b]thiophene-2-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid (Compound 6)

[0194]

[0195] The title compound was prepared using procedures similar to thosedescribed in methods A-G. The product was isolated as a white foam (163mg, 38% last step): IR (solid) 1803, 1736, 1674, 1622, 1527, 1417, 1269cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO+TFA) δ 1.20-1.80 (6H, m), 2.00-2.30 (1H,m), 2.39-3.60 (3H, m), 4.20-4.82 (2H, m), 4.98-5.40 (2H, m), 7.42-7.69(2H, m), 7.72-7.94 (1H, m), 7.99-8.20 (1H, m), 8.23-8.70 (1H m); ¹³C NMR(100 MHz, d₆-DMSO+TFA) δ 18.87, 19.07, 23.25, 23.85, 25.88, 31.47,33.03, 33.20, 33.40, 44.18 (CH₂), 46.05, 46.16, 50.78, 50.82, 50.94,51.02, 51.14, 51.26, 51.42, 56.58, 56.94 (CH), 80.08, 81.84, 81.93,82.05, 83.73, 83.82 (CH₂), 102.44, 102.63, 116.52, 116.68, 116.90 (C),120.79, 122.31, 124.80, 125.55, 125.65 (CH), 129.02, 129.24, 129.32,133.75, 135.65, 160.84, 161.00, 168.93, 169.18, 169.39, 170.64, 171.78,172.64, 200.93, 201.26, 201.40; ¹⁹F (376 MHz, d₆-DMSO +TFA) δ −226.55,−226.79, −226.87, −226.97, −229.76, −229.88, −230.67, −231.15, −232.35,−232.50, −232.56, −232.61; MS (LR, ES) calculated for C₂₀H₂₀ClFN₂O₅S:454.9082, ES−453.296, ES+455.12.

Example 7 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(3-chloro-thiophene-2-carbonyl)-2-piperidinecarboxamido]-pentanoicAcid (Compound 7)

[0196]

[0197] The title compound was prepared using procedures similar to thosedescribed in methods H-J, F and K. The product was isolated as a whitefoam: IR (solid) 1784, 1736, 1670, 1612, 1522, 1450, 1269 cm⁻¹; ¹H NMR(400 MHz, CDCl₃) δ 1.13-1.80 (5H, m), 2.01-3.60 (5H, m), 4.11-5.36 (4H,m), 7.02-7.17 (1H, m), 7.70-7.90 (1H, m), 8.40-8.61 (1H, m); ¹⁹F (376MHz, CDCl₃) δ −226.90 (t), −232.65 (m); Low Res. MS (ES) calculated forC₁₆H₁₈ClFN₂O₅S: 404.8477, ES−403.23, ES+405.062.

Example 8 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-(benzofuran-2-carbonyl)-2-piperidinecarboxamido]pentanoicAcid (Compound 8)

[0198]

[0199] The title compound was prepared using procedures similar to thosedescribed in methods A-G. The product was isolated as a white foam (3.7mg, 6% last step): ¹H NMR (400 MHz, DMSO+TFA) δ 0.70-1.80 (5H, m),2.10-3.60 (5H, m), 4.03-5.40 (4H, m), 7.20-7.83 (5H, m), 8.35-8.80 (1H,m); ¹⁹F (376 MHz, CDCl₃) δ −226.75, −226.89, −232.71; Low Res. MS (ES)calculated for C₂₀H₂₁CFN₂O₆: 404.3986, ES−403.359, ES+405.165.

Example 9 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-[2-(3-trifluoromethyl-phenyl)-thiazole-4-carbonyl]-2-piperidinecarboxamido]-pentanoicAcid (Compound 9)

[0200]

[0201] The title compound was prepared using procedures similar to thosedescribed in methods A-G. The product was isolated as a white foam (31mg, 11% last step): IR (solid) 1789, 1741, 1617, 1512, 1441, 1417, 1327,1231, 1169, 1122 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO+TFA) δ 1.19-1.81 (5H,m), 2.04-3.55 (5H, m, Asp), 4.04-5.39 (4H, m), 7.64-8.36 (5H, m),8.42-8.62 (1H, m); ¹³C NMR (100 MHz, d₆-DMSO+TFA) δ 20.54, 24.71, 25.13,27.12, 27.88, 33.06, 34.54, 34.67, 45.29, 47.62, 52.28, 52.42, 53.09,57.78, 83.22, 83.44, 85.22, 103.92, 104.11, 122.90, 125.50, 125.85,127.35, 128.21, 129.95, 130.26, 130.58, 130.78, 131.07, 133.77, 151.09,163.56, 163.97, 165.03, 171.12, 171.28, 172.10, 172.17, 202.81; ¹⁹F (376MHz, d₆-DMSO) δ −61.76, −226.75, −226.85, −226.96, −227.04, −230.23,−230.35, −230.85, −232.49, −232.6, −232.64, −232.83; MS (LR, ES)calculated for C₂₂H₂₁F₄N₃O₅S: 515.4874, ES−514.361, ES+516.167.

Example 10 [3S/R,(2S)]-5-Fluoro-4-oxo-3-[1-[2-(3-trifluoromethyl-phenyl)-furan-4-carbonyl]-2-piperidinecarboxamido]-pentanoicAcid (Compound 10)

[0202]

[0203] The title compound was prepared using procedures similar to thosedescribed in methods A-G. The product was isolated as a white foam (82mg, 18% last step): IR (solid) 1794, 1736, 1670, 1603, 1522, 1431, 1331,1255, 1165 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO +TFA) δ 1.25-3.58 (10H, m),4.18-5.34 (4H, m), 6.98-7.41 (2H, m), 7.62-7.78 (2H, m), 7.91-8.13 (2H,m), 8.10-8.80 (1H, m); ¹³ C NMR (100 MHz, d₆-DMSO +TFA) δ 19.18, 19.42,31.48, 33.13, 33.32, 50.87, 50.96, 81.95, 81.98, 83.73, 83.76, 102.49,102.54, 102.69, 107.73, 116.84, 118.78, 119.28, 121.49, 123.66, 124.20,126.62, 126.91, 128.68, 129.00, 129.16, 129.21, 129.31, 145.80, 145.87,151.28, 151.39, 157.83, 158.40, 169.27, 169.64, 170.60, 170.70, 171.80,201.16, 201.30, 201.43; ¹⁹F (376 MHz, d₆-DMSO +TFA) δ −61.63, −226.79,−232.56; MS (LR, ES) calculated for C₂₃H₂₂F₄N₂O₆: 498.4352, ES−497.313,ES+499.233.

Example 11 [3S/R,(2S)]-5-Fluoro-3-{[1-(3-methyl-5-phenyl-thiophene-2-carbonyl)piperidine-2-carbonyl]-amino}-4-oxo-pentanoic Acid (Compound 11)

[0204]

[0205] Method N: 5-Bromo-3-methyl-thiophene-2-carbaldehyde

[0206] A solution of 3-methylthiophene-2-carbaldehyde (10 g, 0.079 mol)in dichloromethane (10 ml) was added dropwise to a stirred solution ofbromine (4.08 ml, 0.079 mol) in dichloromethane (15 ml) at roomtemperature. The resulting mixture was heated to reflux temperature for3 hours before cooling to room temperature, washed with water (3×50 ml),saturated NaHCO₃ solution (2×25 ml), dried (MgSO₄) and the solventremoved at reduced pressure to give the sub-title compound as a brownsolid (14.7 g, 66% yield): ¹H NMR (400 MHz, CDCl₃) δ_(H) 2.60 (3H, s),6.97 (1H, s) 9.20 (1H, s).

[0207] Method O: 3-Methyl-5-phenyl-thiophene-2-carbaldehyde

[0208] To a solution of 5-bromo-3-methyl-thiophene-2-carbaldehyde (1.00g, 4.88 mmol) in ethylene glycol dimethyl ether (9 ml) was addedphenylboronic acid (0.773 g, 6.34 mmol), 2M Na₂CO₃ solution (6.3 ml) andPd(PPh₃)₄ [0.282 g, 0.24 mmol). The mixture was heated for 18 hours,cooled and the solvent removed at reduced pressure to leave a brownresidue which was partitioned between water (15 ml) and dichloromethane(20 ml). The organic was separated, washed with water (2×5 ml), brine(10 ml), dried (MgSO₄) and the solvent removed at reduced pressure togive a brown oil. Purification by flash column chromatography (6:1petrol 40-60° C./ethyl acetate) gave the sub-title compound as a yellowoil (0.90 g, 91% yield): ¹H NMR (400 MHz, CDCl₃) δ_(H) 2.75 (3H, s),7.25 (1H, s) 7.35-7.75 (5H, s), 10.05 (1H, s).

[0209] Method P: 3-Methyl-5-phenyl-thiophene-2-carboxylic Acid

[0210] To a stirred solution of3-methyl-5-phenyl-thiophene-2-carbaldehyde (0.200 g, 0.99 mmol) and2-methyl-2-butene (2.77 g, 0.040 mol) in dimethylformamide (4 ml) at 0°C. was added NaClO₂ (0.894 g, 9.89 mmol) and NaH₂PO₄ (1.09 g, 7.91 mmol)in water (5 ml). The solution was allowed to warm to room temperatureand stirred for 18 hours. The solvent was removed at reduced pressureand the residue partitioned between dichloromethane (10 ml) and 1N HClsolution (10 ml). The organic was separated, the aqueous layer extractedwith dichloromethane (2×5 ml). The combined organic layers were dried(MgSO₄) and the solvent removed at reduced pressure to give a yellowoil. Purification by flash column chromatography 50% ethylacetate/petrol 40-60° C.) gave the sub-title compound as a white solid(0.14 g, 69% yield): ¹H NMR (400 MHz, CDCl₃) δ_(H) 2.65 (3H, s), 7.25(1H, s) 7.35-7.80 (5H, s).

[0211] Method Q: [3S/R,(2S)]-5-Fluoro-3-{[1-(3-methyl-5-phenyl-thiophene-2-carbonyl)piperidine-2-carbonyl]-amino}-4-oxo-pentanoic Acid

[0212] The title compound was prepared from3-methyl-5-phenyl-thiophene-2-carboxylic acid using procedures similarto those described in Methods F, H-K. The product was isolated as awhite foam (0.066 g, 81% yield): IR (film) 1781.5, 1715.3, 1668.0,1597.1, 1441.0, 1190.3 cm-1; ¹H NMR (400 MHz, DMSO) δ_(H) 1.00-1.80 (6H,m), 1.90-2.30 (4H, m), 2.70-3.90 (4H, m), 4.10-5.50 (4H, 2×m), 7.30-8.60(6H, 4×m); ¹⁹F NMR (376 MHz, DMSO) −61.7, −224.3, −226.7, −226.8,−227.5, −232.7, −233.4; MS (FAB+ve, HR) Calculated for C₂₃H₂₄FN₂O₅S(MH−) 459.52, found 459.40.

Example 12 [3S/R,(2S)]-5-Fluoro-3-({1-[3-methyl-5-(3-trifluoromethyl-phenyl)-thiophene-2-carbonyl]-piperidine-2-carbonyl}-amino)-4-oxo-pentanoicAcid (Compound 12)

[0213]

[0214] The title compound was prepared from3-methylthiophene-2-carbaldehyde using procedures similar to thosedescribed in Methods F, H-K, N-P. The product was isolated as a palepink solid (0.16 g, 94%): IR (film) 1784.0, 1726.9, 1664.9, 1588.7,1436.2, 1326.6, 1164.6 cm-1; ¹H NMR (400 MHz, DMSO) δ_(H) 1.00-1.80 (6H,m), 1.90-2.30 (4H, m), 2.70-4.05 (4H, m), 4.10-5.40 (4H, m), 7.00-9.00(6H, m); ¹⁹F NMR (376 MHz, DMSO) −62.0, −224.3, −226.7, −226.9, −227.5,−232.6, −232.7, −233.4; MS (FAB+ve, HR) Calculated for C₂₄H₂₃F₄N₂O₅S(MH−) 527.41, found 527.52.

Example 13 [3S/R,(2S)]-5-Fluoro-4-oxo-3-({1-[5-(3-trifluoromethyl-phenyl)-thiophene-2-carbonyl]-piperidine-2S-carbonyl}-amino)-pentanoicAcid (Compound 13)

[0215]

[0216] The title compound was prepared from thiophene-2-carbaldehydeusing procedures similar to those described in Methods F, H-K, N-P. Theproduct was isolated as a pale yellow solid (0.23 g, 93%): IR (film)1784.0, 1722.1, 1664.9, 1588.7, 1531.5, 1321.8, 1164.6 cm-1; ¹H NMR (400MHz, DMSO) δ_(H) 0.90-1.85 (6H, m), 2.00-2.40 (1H, m), 2.45-3.50 (3H,m), 3.90-5.55 (4H, m), 7.00-9.05 (7H, m); ¹⁹F NMR (376 MHz, DMSO) −61.7,−224.3, −226.7, −226.8, −227.5, −227.6, −232.7, −233.4; MS (FAB+ve, HR)Calculated for C₂₃H₂₃F₄N₂O₅S (MH+) 515.51, found 515.35.

Example 14 [3S/R,(2S)]-5-Fluoro-4-oxo-3-{[1-(pyridine-2-carbonyl)-piperidine-2-carbonyl]-amino}-pentanoicAcid (Compound 14)

[0217]

[0218] The title compound was prepared from pyridine-2-carboxylic acidusing procedures similar to those described in Methods F, H-K. Theproduct was isolated as a white solid (0.10 g, 93%):IR (film) 2945.4,1650.5, 1446.6, 1186.5, 1139.9 cm⁻¹; ¹H NMR (400 MHZ, CDCl₃) δ_(H)1.40-1.80 (6H, m), 2.20-2.50 (1H, m), 2.69-3.12 (2H, m), 3.29-3.35 (1H,m), 3.48-3.51 (1H, m), 4.47-5.29 (3H, m), 7.37-9.11 (6H, m); ¹⁹F NMR(376 MHz, CDCl₃) −231.69, −231.56, −231.44; MS (FAB+ve, HR) Calculatedfor C₁₇H₂₁FN₃O₅ (MH+) 366.36, found 366.4.

Example 15 [3S/R,(2S)]-3-{[1-(Biphenyl-3-carbonyl)-piperidine-2-carbonyl]-amino}-5-fluoro-4-oxo-pentanoicAcid (Compound 15)

[0219]

[0220] The title compound was prepared from 3-biphenylcarboxylic acidusing procedures similar to those described in Methods F, H-K. Theproduct was isolated as a white solid (0.13 g, 97%): IR (film) 2930.9,1782.2, 1723.7, 1668.6, 1596.1, 1444.2, 1174.7 cm-1; ¹H NMR (400 MHz,CDCl₃) δ_(H) 1.40-1.90 (6H, m), 2.19-2.41 (1H, m), 2.70-3.30 (3H, m),3.70-3.85 (1H, m), 4.30-5.50 (3H, m), 7.35-7.70 (11H, m); ¹⁹F NMR (376MHz, CDCl₃) −229.60, −229.88; MS (FAB+ve, HR) Calculated for C₂₄H₂₆FN₂O₅(MH+) 441.46, found 441.4.

Biological Methods Example 16 Enzyme Assays

[0221] The assays for caspase inhibition are based on the cleavage of afluorogenic substrate by recombinant, purified human Caspases-1, -3, -7or -8. The assays are run in essentially the same way as those reportedin WO01/42216.

[0222] The compounds of examples 1-15 possess K_(inact)values>5,000M⁻¹s⁻¹ against caspases-1, -3 and -8.

Example 17 Inhibition of IL-1β secretion from Mixed Population ofPeripheral Blood Mononuclear Cells (PBMC)

[0223] Processing of pre-IL-1β by caspase-1 can be measured in cellculture using a variety of cell sources. Human PBMC obtained fromhealthy donors provides a mixed population of lymphocyte and mononuclearcells that produce a spectrum of interleukins and cytokines in responseto many classes of physiological stimulators. The assay conditions usedfor inhibition of IL-1β secretion from mixed population of peripheralblood mononuclear cells can be found in WO01/42216.

[0224] The inhibitory potency of the compounds can be represented by anIC₅₀ value, which is the concentration of inhibitor at which 50% of themature IL-1β is detected in the supernatant as compared to the positivecontrols. Compound 10 of this invention showed an IC₅₀ of less than 0.5μM in inhibition of IL-1β secretion from peripheral blood mononuclearcells as determined by the above methods.

Example 18 Anti-Fas Induced Apoptosis Assay

[0225] Cellular apoptosis can be induced by the binding of Fas ligand(FasL) to its receptor, CD95 (Fas). Conditions for an assay to measurethe effect of compounds on the inhibition of the caspase-8-mediatedapoptotic pathway can be found in WO01/42216.

[0226] Compound 3 of this invention showed an IC₅₀ of less than 0.05 μMin the FAS induced apoptosis assay.

Example 19 Inhibition of TNF Release from Whole Blood

[0227] Human blood was freshly drawn from healthy donors and collectedin vacutainers. Blood was diluted 1:2 in PBS (tissue culture, pyrogenfree) in a sterile bottle and inverted to mix well. Aliquots of 0.5 mlof blood mixture were dispensed into cluster tubes in 96 well format.

[0228] Dilutions of the test compounds were prepared in RPMI by taking100 mM DMSO stocks of the compounds and diluting 1:10 in RPMI medium ineppendorfs, to give a 10 mM stock. 1:5 serial dilutions were preparedfrom the stock solutions.

[0229] LPS was kept at a frozen stock (−20 degrees C.) at 1 mg/ml in PBSand then diluted to 1:10 with RPMI medium and finally diluted in themedium again 1:350. 501l of each test compound (first concentration was100uM) were added to the blood samples and then stimulated with 10 μlLPS (final concentration in the well is 5 ng/ml). The contents weregently mixed using an 8 well multi-channel pipette and incubated at 37°C. over night. At the end of the incubation time, contents were gentlymixed, then spun down at 1000× g for 5 mins at 20° C. The serumsupernatants were transferred to a fresh plate without disturbing theRBCs and diluted 1:2 with the diluent RD6C.

[0230] TNF-alpha levels of supernatants were assayed using the R+Dsystems ELISA kit, using R+D systems protocol. Samples were read at 450nm. Most preferred compounds of this invention showed IC₅₀ of less than6 μM in the LPS-induced TNF-alpha assay in whole blood.

[0231] Compound 10 of this invention showed an IC₅₀ of less than 6 μM(5044 nM) in the LPS induced TNF-alpha assay in whole blood.

[0232] While we have described a number of embodiments of thisinvention, it is apparent that our basic examples can be altered toprovide other embodiments, which utilize the compounds and methods ofthis invention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments, which have been represented by way of example.

We claim:
 1. A compound of the formula I:

wherein: R¹ is hydrogen, CN, CHN₂, R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, an aryl group, a substituted aryl group, an aralkyl group, a substituted aralkyl group, a non-aromatic heterocyclic group, or a substituted non-aromatic heterocyclic group; Y is an electronegative leaving group, —OR, —SR, —OC═O(R), or —OPO(R³) (R⁴); R³ and R⁴ are independently R or OR; R² is CO₂H, CH₂CO₂H, or optionally substituted esters, amides or isosteres thereof; A is C═O or SO₂; X¹ is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionally substituted by an alkyl group, a cycloalkyl group, a (cycloalkyl)alkyl group, an amino acid N-terminal protecting group, or COR and —CH₂ is optionally substituted by fluorine, an alkyl group, a cycloalkyl group, a (cycloalkyl)alkyl group, an aralkyl group, an aryl group, an alkyloxy group, an alkylthioxy group, an aryloxy group, an arylthioxy group, an oxo group (i.e., ═O), or a NHCOR group; X² is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionally substituted by an alkyl group, or an amino acid N-terminal protecting group and —CH₂ is optionally substituted by an alkyl group, an aryl group, an alkyloxy group, an alkylthioxy group, an aryloxy group, an arylthioxy group, or an oxo (i.e., ═O) group, a NHCOR group; X¹ and X² optionally form part of a phenyl ring that is fused to the adjoining ring Q; X³ is CH₂ or X² and X³ optionally form part of a phenyl ring that is fused to the adjoining ring Q, provided that when X² forms a ring with X³ then X² does not form a ring with X¹; any two hydrogens attached to adjacent positions in ring Q are optionally replaced by a double bond; and Z is an optionally substituted ring selected from the group consisting of a carbocyclic, an aryl, a saturated heterocycle, a partially saturated heterocycle, and a heteroaryl wherein the ring is connected to A at a ring carbon; or a pharmaceutically acceptable derivative thereof.
 2. The compound of claim 1 wherein R¹ is CH₂Y and Y is F, OR, SR, or —OC(═O) (R).
 3. The compound of claim 2 wherein Y is F.
 4. The compound of claim 2 wherein R² is CO₂H, an ester, amide, or carboxylic acid isostere.
 5. The compound of claim 4 wherein R² is CO₂H.
 6. The compound of claim 4 wherein X¹ and X² are each CH₂, or X¹ and X² combine to form part of an optionally substituted phenyl ring fused to ring Q.
 7. The compound of claim 6 wherein X¹ and X² are each CH₂.
 8. The compound of claim 7 wherein A is CO.
 9. The compound of claim 8 wherein Z is an optionally substituted aryl which is connected to A at a ring carbon.
 10. The compound of claim 1 selected from Table 1 below: TABLE 1 Representative Compounds

No. Z 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25


11. A pharmaceutical composition comprising: a) a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10; and b) a pharmaceutically acceptable carrier, adjuvant or vehicle.
 12. A method for treating or preventing a disease selected from a group consisting of an IL-1 mediated disease, an apoptosis mediated disease, a TNF-alpha mediated disease, an inflammatory disease, an autoimmune disease, a destructive bone disorder, a proliferative disorder, an infectious disease, a degenerative disease, a skin disease, a disease associated with cell death, an excess dietary alcohol intake disease, a viral mediated disease, retinal disorder, uveitis, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, psoriasis, atopic dermatitis, contact dermatitis, scarring, graft vs host disease, organ transplant rejection, organ apoptosis after burn injury, osteoporosis, leukemias and related disorders, myelodysplastic syndrome, multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma, haemorrhagic shock, sepsis, septic shock, burns, trauma, systemic inflammatory response syndrome, multiple organ dysfunction syndrome, Shigellosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Kennedy's disease, prion disease, cerebral ischemia, epilepsy, myocardial ischemia, acute and chronic heart disease, myocardial infarction, congestive heart failure, atherosclerosis, coronary artery bypass graft, spinal muscular atrophy, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related encephalitis, aging, alopecia, neurological damage due to stroke, ulcerative colitis, traumatic brain injury, spinal chord injury, hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, Japanese encephalitis, various forms of liver disease, renal disease, polycystic kidney disease, H. pylori-associated gastric and duodenal ulcer disease, HIV infection, tuberculosis, and meningitis in a subject comprising the step of administering to said subject a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 13. The method according to claim 12, wherein the disease is an apoptosis mediated disease, an inflammatory disease, an autoimmune disease, a destructive bone disorder, a proliferative disorder, an infectious disease, a degenerative disease, a disease associated with cell death, an excess dietary alcohol intake disease, a viral mediated disease, inflammatory peritonitis, glomerulonephritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, scarring, graft vs host disease, organ transplant rejection, osteoporosis, leukemias and related disorders, myelodysplastic syndrome, metastatic melanoma, haemorrhagic shock, sepsis, septic shock, burns, trauma, systemic inflammatory response syndrome, multiple organ dysfunction syndrome, Shigellosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Kennedy's disease, prion disease, cerebral ischemia, epilepsy, myocardial ischemia, acute and chronic heart disease, myocardial infarction, congestive heart failure, atherosclerosis, coronary artery bypass graft, spinal muscular atrophy, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related encephalitis, aging, alopecia, neurological damage due to stroke, traumatic brain injury, spinal chord injury, hepatitis-B, hepatitis-C, hepatitis-G, various forms of liver disease, renal disease, polycystic kidney disease, H. pylori-associated gastric and duodenal ulcer disease, HIV infection, tuberculosis, or meningitis.
 14. A method for inhibiting a caspase-mediated function in a subject comprising the step of administering to said subject a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 15. A method for decreasing TNF-alpha levels or activity in a subject comprising the step of administering to said subject a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 16. A method for decreasing IGIF- or IFN-γ production in a subject comprising the step of administering to said subject a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 17. A method for treating complications associated with coronary artery bypass grafts comprising the step of administering to said subject a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 18. A method for preserving cells comprising the step of bathing the cells in a solution of a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10.
 19. A method according to claim 18, wherein said compound or pharmaceutically acceptable derivative thereof is used for an organ transplant or for preserving blood products.
 20. A method of treating cancer comprising the step of administering a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 wherein said compound or composition is used as a component of immunotherapy.
 21. The method according to any one of claims 12-19 wherein said compound, derivative or composition is administered with an additional therapeutic agent.
 22. The method of claim 21 wherein said additional therapeutic agent is selected from a group consisting of a thrombolytic agent, an anti-inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin, and an anti-vascular hyper-proliferation compound.
 23. A method for inhibiting TNF-mediated conditions in a subject comprising the step of administering to said subject a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 24. The method according to claim 23, wherein said TNF-mediated conditions are selected from a group consisting of restinosis, inflammatory diseases of the central nervous system, demyelinating diseases of the nervous system, multiple sclerosis, septic arthritis, aneurysmal aortic disease, traumatic joint injury, peridontal disease, macular degeneration, diabetic retinopathy, occular inflammation, keratoconus, Sjogren's syndrome, corneal graft rejection, cachexia, and anorexia.
 25. A method for identifying a compound that decreases TNF-alpha levels in a cell culture comprising the steps of administering a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 to the cell culture and comparing the amount of TNF-alpha present to the amount of TNF-alpha present in a cell culture that has not been treated with the compound.
 26. A method for identifying a compound that decreases TNF-alpha activity in a cell culture comprising the steps of administering a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 to the cell culture and comparing the amount of TNF-alpha present to the amount of TNF-alpha present in a cell culture that has not been treated with the compound.
 27. A method for decreasing TNF-alpha levels or activity in a cell culture comprising the step of administering to the cell culture a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim
 11. 28. A kit comprising a caspase inhibitor and a tool for measuring TNF-alpha levels or activity.
 29. A method for identifying a compound for decreasing TNF-alpha levels in a subject comprising administering a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1 to 10 or a pharmaceutical composition comprising the compound and comparing the TNF-alpha levels present in the subject before and after treatment with the compound.
 30. A method for identifying a compound for decreasing TNF-alpha activity in a subject comprising administering a compound or a pharmaceutically acceptable derivative thereof according to any one of claims 1 to 10 or a pharmaceutical composition comprising the compound and comparing the TNF-alpha activity present in the subject before and after treatment with the compound. 